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Sample records for cell penetrating peptide

  1. Cell Penetration Properties of a Highly Efficient Mini Maurocalcine Peptide

    Directory of Open Access Journals (Sweden)

    Michel De Waard

    2013-03-01

    Full Text Available Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin’s hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments.

  2. Prediction of cell-penetrating peptides with feature selection techniques.

    Science.gov (United States)

    Tang, Hua; Su, Zhen-Dong; Wei, Huan-Huan; Chen, Wei; Lin, Hao

    2016-08-12

    Cell-penetrating peptides are a group of peptides which can transport different types of cargo molecules such as drugs across plasma membrane and have been applied in the treatment of various diseases. Thus, the accurate prediction of cell-penetrating peptides with bioinformatics methods will accelerate the development of drug delivery systems. The study aims to develop a powerful model to accurately identify cell-penetrating peptides. At first, the peptides were translated into a set of vectors with the same dimension by using dipeptide compositions. Secondly, the Analysis of Variance-based technique was used to reduce the dimension of the vector and explore the optimized features. Finally, the support vector machine was utilized to discriminate cell-penetrating peptides from non-cell-penetrating peptides. The five-fold cross-validated results showed that our proposed method could achieve an overall prediction accuracy of 83.6%. Based on the proposed model, we constructed a free webserver called C2Pred (http://lin.uestc.edu.cn/server/C2Pred). PMID:27291150

  3. Prediction of cell penetrating peptides by support vector machines.

    Directory of Open Access Journals (Sweden)

    William S Sanders

    2011-07-01

    Full Text Available Cell penetrating peptides (CPPs are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs. We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating.

  4. Strategies to stabilize cell penetrating peptides for in vivo applications.

    Science.gov (United States)

    Fominaya, Jesús; Bravo, Jerónimo; Rebollo, Angelita

    2015-10-01

    In the era of biomedicines and engineered carrier systems, cell penetrating peptides (CPPs) have been established as a promising tool for therapeutic application. Likewise, other therapeutic peptides, successful in vivo application of CPPs will strongly depend on peptide stability, the bottleneck for this type of biodegradable molecules. In this review, the authors describe the current knowledge of the in vivo degradation for known CPPs and the different strategies available to provide a higher resistance to metabolic degradation while preserving cell penetration efficiency. Peptide stability can be improved by different means, either modifying the structure to make it unrecognizable to proteases, or preventing access of proteolytic enzymes by applying conformation restriction or shielding strategies. PMID:26448473

  5. Cell-penetrating peptides: breaking through to the other side.

    Science.gov (United States)

    Koren, Erez; Torchilin, Vladimir P

    2012-07-01

    Cell-penetrating peptides (CPPs) have been previously shown to be powerful transport vector tools for the intracellular delivery of a large variety of cargoes through the cell membrane. Intracellular delivery of plasmid DNA (pDNA), oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, drugs, as well as various nanoparticulate pharmaceutical carriers (e.g., liposomes, micelles) has been demonstrated both in vitro and in vivo. This review focuses on the peptide-based strategy for intracellular delivery of CPP-modified nanocarriers to deliver small molecule drugs or DNA. In addition, we discuss the rationales for the design of 'smart' pharmaceutical nanocarriers in which the cell-penetrating properties are hidden until triggered by exposure to appropriate environmental conditions (e.g., a particular pH, temperature, or enzyme level), applied local microwave, ultrasound, or radiofrequency radiation. PMID:22682515

  6. Intracellular Delivery of Nanoparticles with Cell Penetrating Peptides.

    Science.gov (United States)

    Salzano, Giuseppina; Torchilin, Vladimir P

    2015-01-01

    The functionalization of nanoparticles (NPs) with cell penetrating peptides (CPPs) constitutes a breakthrough for the intracellular delivery of therapeutic and diagnostic payloads. In late 1998, a significant cellular uptake of a small protein from the HIV-1 virus, namely TAT peptide (TATp), was observed. Thereafter, research began on design of similarly acting peptides, and the coupling of NPs with these novel CPPs. Here, we describe recent methods used to modify the surface of NPs with CPPs and the in vitro and in vivo effects of such functionalization on the intracellular delivery of various cargos. In particular, we highlight recent advances aimed at reducing the non-selectivity of CPPs and the prevention of their enzymatic cleavage en route to target tissues. PMID:26202282

  7. Peptide Internalization Enabled by Folding: Triple Helical Cell-Penetrating Peptides

    OpenAIRE

    Shinde, Aparna; Feher, Katie M.; Hu, Chloe; Slowinska, Katarzyna

    2014-01-01

    Cell-Penetrating Peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degrada...

  8. Tumor penetrating peptides

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    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  9. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    Science.gov (United States)

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

  10. Alternative Mechanisms for the Interaction of the Cell-Penetrating Peptides Penetratin and the TAT Peptide with Lipid Bilayers

    NARCIS (Netherlands)

    Yesylevskyy, Semen; Marrink, Siewert-Jan; Mark, Alan E.

    2009-01-01

    Cell-penetrating peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with 1,2-Dipa

  11. Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

    DEFF Research Database (Denmark)

    Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J;

    2015-01-01

    La WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition...

  12. Angiopep-2 and activatable cell penetrating peptide dual modified nanoparticles for enhanced tumor targeting and penetrating.

    Science.gov (United States)

    Mei, Ling; Zhang, Qianyu; Yang, Yuting; He, Qin; Gao, Huile

    2014-10-20

    Delivering chemotherapeutics by nanoparticles into tumor was influenced by at least two factors: specific targeting and highly efficient penetrating of the nanoparticles. In this study, two targeting ligands, angiopep-2 and activatable cell penetrating peptide (ACP), were functionalized onto nanoparticles for tumor targeting delivery. In this system, angiopep-2 is a ligand of low-density lipoprotein receptor-related protein-1 (LRP1) which was highly expressed on tumor cells, and the ACP was constructed by the conjugation of RRRRRRRR (R8) with EEEEEEEE through a matrix metalloproteinase-2 (MMP-2) sensitive linker, enabling the ACP with tumor microenvironment-responsive cell penetrating property. 4h incubation of ACP with MMP-2 leads to over 80% cleavage of ACP, demonstrating ACP indeed possessed MMP-2 responsive property. The constructed dual targeting nanoparticles (AnACNPs) were approximately 110 nm with a polydispersity index of 0.231. In vitro, ACP modification and angiopep-2 modification could both enhance the U-87 MG cell uptake because of the high expression of MMP-2 and LRP-1 on C6 cells. AnACNPs showed higher uptake level than the single ligand modified nanoparticles. The uptake of all particles was time- and concentration-dependent and endosomes were involved. In vivo, AnACNPs showed best tumor targeting efficiency. The distribution of AnACNPs in tumor was higher than all the other particles. After microvessel staining with anti-CD31 antibody, the fluorescent distribution demonstrated AnACNPs could distribute in the whole tumor with the highest intensity. In conclusion, a novel drug delivery system was developed for enhanced tumor dual targeting and elevated cell internalization.

  13. Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone

    DEFF Research Database (Denmark)

    Jing, Xiaona; Yang, Mingjun; Kasimova, Marina Robertovna;

    2012-01-01

    Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone were studied...

  14. Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8

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    Gautam, Ankur; Nanda, Jagpreet Singh; Samuel, Jesse S.; Kumari, Manisha; Priyanka, Priyanka; Bedi, Gursimran; Nath, Samir K.; Mittal, Garima; Khatri, Neeraj; Raghava, Gajendra Pal Singh

    2016-01-01

    Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications. PMID:27189051

  15. Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8.

    Science.gov (United States)

    Gautam, Ankur; Nanda, Jagpreet Singh; Samuel, Jesse S; Kumari, Manisha; Priyanka, Priyanka; Bedi, Gursimran; Nath, Samir K; Mittal, Garima; Khatri, Neeraj; Raghava, Gajendra Pal Singh

    2016-01-01

    Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications. PMID:27189051

  16. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria.

    Science.gov (United States)

    Jain, Aastha; Chugh, Archana

    2016-09-01

    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  17. Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake.

    NARCIS (Netherlands)

    Verdurmen, W.P.R.; Thanos, M.; Ruttekolk, I.R.R.; Gulbins, E.; Brock, R.E.

    2010-01-01

    Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and - at higher peptide concentrations - in an endocytosis-independent manner via localized regions of the plasm

  18. A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency.

    NARCIS (Netherlands)

    Duchardt, F.; Ruttekolk, I.R.R.; Verdurmen, W.P.R.; Lortat-Jacob, H.; Burck, J.; Hufnagel, H.; Fischer, R.; Heuvel, M. van den; Lowik, D.W.; Vuister, G.W.; Ulrich, A.; Waard, M. de; Brock, R.E.

    2009-01-01

    The molecular events that contribute to the cellular uptake of cell-penetrating peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a 22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformati

  19. Application of Cell Penetrating Peptide in Magnetic Resonance Imaging of Bone Marrow Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Min LIU; You-Min GUO; Jun-Le YANG; Peng WANG; Lin-Yu ZHAO; Nian SHEN; Si-Cen WANG; Xiao-Juan GUO; Qi-Fei WU

    2006-01-01

    Tracking the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs).MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro. The cellpenetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by a solid-phase peptide synthesis method. Fluorescein imaging analysis confirmed that this new peptide could internalize into the cytoplasm and nucleus at room temperature, 4℃ and 37℃. Gadolinium were efficiently internalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner,resulting in intercellular shortening of longitudinal relaxation enhancements, which were obviously detected by 1.5 Tesla Magnetic Resonance Imaging. Cytotoxicity assay and flow cytometric analysis showed that the intercellular contrast medium incorporation did not affect cell viability at the tested concentrations. The in vitro experiment results suggested that the new constructed peptides could be a vector for tracking MSCs.

  20. Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

    Directory of Open Access Journals (Sweden)

    Armelle Tchoumi Neree

    2015-11-01

    Full Text Available Over the last two decades, the potential usage of cell-penetrating peptides (CPPs for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38 has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs. Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

  1. Oligonucleotide delivery with cell surface binding and cell penetrating Peptide amphiphile nanospheres.

    Science.gov (United States)

    Mumcuoglu, Didem; Sardan, Melis; Tekinay, Turgay; Guler, Mustafa O; Tekinay, Ayse B

    2015-05-01

    A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonucleotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonucleotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R4 and R8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R8-PA and KRSR-PA. R8 and R8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs. PMID:25828697

  2. Methods to Study the Role of the Glycocalyx in the Uptake of Cell-Penetrating Peptides

    NARCIS (Netherlands)

    Schmidt, S.; Wallbrecher, R.; Kuppevelt, T.H. van; Brock, R.E.

    2015-01-01

    Cells are covered by a layer of negatively charged oligo- and polysaccharides, the glycocalyx. Cell-penetrating peptides and other drug delivery vehicles first encounter these polyanions before contacting the lipid bilayer of the plasma membrane. While a large body of data supports the notion that i

  3. Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide

    DEFF Research Database (Denmark)

    Abdul, Fabien; Ndeboko, Bénédicte; Buronfosse, Thierry;

    2012-01-01

    of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)8 having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion......Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication...

  4. Conformational analysis of Infectious bursal disease virus (IBDV derived cell penetrating peptide (CPP analogs

    Directory of Open Access Journals (Sweden)

    Vinay G. Joshi

    2013-12-01

    Full Text Available Aim: This study was designed to develop peptide analogs of Infectious Bursal Disease (IBD virus VP5 protein segment having cell penetrating ability to improve their interaction with cargo molecule (Nucleic acid without affecting the backbone conformation. Materials and Methods: IBDV VP5 protein segment designated as RATH peptide were synthesized using solid phase peptide synthesis and their solution conformation was elucidated using CD spectroscopy in polar (water and apolar (TFE solvents. Cell penetrating ability of RATH-CONH2 was observed using FITC labeled peptide internalization in to HeLa cells under fluorescent microscopy. The efficacy of RATH analog interactions with nucleic acids was evaluated using FITC labeled oligonucleotides by fluorescence spectroscopy and plasmid constructs in gel retardation assay. Results: CD spectra of RATH analogs in water and apolar trifluroethanol (TFE helped to compare their secondary structures which were almost similar with dominant beta conformations suggesting successful induction of positive charge in the analogs without affecting back bone conformation of CPP designed. Cell penetrating ability of RATH CONH2 in HeLa cell was more than 90%. The fluorescence spectroscopy and plasmid constructs in gel retardation assay demonstrated successful interaction of amide analogs with nucleic acid. Conclusion: Intentional changes made in IBDV derived peptide RATH COOH to RATH CONH2 did not showed major changes in backbone conformation and such modifications may help to improve the cationic charge in most CPPs to interact with nucleic acid. [Vet World 2013; 6(6.000: 307-312

  5. Polymeric pH nanosensor with extended measurement range bearing octaarginine as cell penetrating peptide

    DEFF Research Database (Denmark)

    Ke, Peng; Sun, Honghao; Liu, Mingxing;

    2016-01-01

    A synthetic peptide octaarginine which mimics human immunodeficiency virus-1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental pH-s...

  6. Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

    DEFF Research Database (Denmark)

    Kristensen, Mie; Birch, Ditlev; Mørck Nielsen, Hanne

    2016-01-01

    -penetrating peptides (CPPs) constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB). CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide...... or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific...... barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate...

  7. Peptide based DNA nanocarriers incorporating a cell-penetrating peptide derived from neurturin protein and poly-L-lysine dendrons.

    Science.gov (United States)

    Rosli, Nurlina; Christie, Michelle P; Moyle, Peter M; Toth, Istvan

    2015-05-15

    Multicomponent gene delivery systems incorporating cell-penetrating peptides (CPP) from the human neurturin protein (NRTN-30, NRTN(132-161); NRTN-17, NRTN(145-161)) and a poly-l-lysine (PLL) dendron, were synthesized and characterized for plasmid DNA (pDNA) delivery. Acetylated NRTN peptides (Ac-CPP) and peptides conjugated to a PLL dendron (DEN-CPP) efficiently condensed and stabilized pDNA. Complexes between pDNA and DEN-CPP formed smaller and more stable nanoparticles. Flow cytometry experiments showed that pDNA-DEN-CPPs were taken up more efficiently into HeLa cells. There was also no significant difference between NRTN-30 and NRTN-17 for pDNA uptake, indicating that the truncated peptide alone is sufficient as a CPP for pDNA delivery.

  8. A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity.

    Science.gov (United States)

    Ru, Qin; Shang, Bo-Yang; Miao, Qing-Fang; Li, Liang; Wu, Shu-Ying; Gao, Rui-Juan; Zhen, Yong-Su

    2012-11-20

    Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs. PMID:22982402

  9. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    Science.gov (United States)

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

  10. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    Science.gov (United States)

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide. PMID:27576711

  11. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

    Science.gov (United States)

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M

    2002-08-01

    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  12. Cell-Penetrating Peptides: A Comparative Study on Lipid Affinity and Cargo Delivery Properties

    Directory of Open Access Journals (Sweden)

    Paolo Ruzza

    2010-03-01

    Full Text Available A growing number of natural and/or synthetic peptides with cell membrane penetrating capability have been identified and described in the past years. These molecules have been considered promising tools for delivering bioactive compounds into various cell types. Although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. In this work we try to connect the capability to interact with model lipid membrane and structural and chemical characteristics of CPPs in order to obtain a biophysical classification that predicts the behavior of CPP-cargo molecules in cell systems. Indeed, the binding with cell membrane is one of the primary step in the interaction of CPPs with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how CPPs may translocate a membrane without destroying it and how this interactions come into play in shuttling CPPs via different routes with different efficiency. We analyzed by CD and fluorescence spectroscopies the binding properties of six different CPPs (kFGF, Nle54-Antp and Tat derived peptides, and oligoarginine peptides containing 6, 8 or 10 residues in absence or presence of the same cargo peptide (the 392-401pTyr396 fragment of HS1 protein. The phospholipid binding properties were correlated to the conformational and chemical characteristics of peptides, as well as to the cell penetrating properties of the CPP-cargo conjugates. Results show that even if certain physico-chemical properties (conformation, positive charge govern CPP capability to interact with the model membrane, these cannot fully explain cell-permeability properties.

  13. Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

    International Nuclear Information System (INIS)

    The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 ± 0.0122 mmol-1 s-1, higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells

  14. SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    ZHAI Xiao-hui; LIU Min; GUO Xiao-juan; WANG Si-cen; ZHANG Hong-xia; GUO You-min

    2011-01-01

    Background The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines,SKOV-3.Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid gadolinium (Ⅲ) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P <0.01) and concentration-dependent manner (r=0.964, P <0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

  15. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck

    2008-01-01

    , proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell...

  16. Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development

    OpenAIRE

    Ezzat, Kariem

    2012-01-01

    Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides ...

  17. Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

    Directory of Open Access Journals (Sweden)

    Caghan Kizil

    Full Text Available Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI, RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

  18. Cell-penetrating recombinant peptides for potential use in agricultural pest control applications.

    Science.gov (United States)

    Hughes, Stephen R; Dowd, Patrick F; Johnson, Eric T

    2012-09-28

    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider (Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.

  19. Cell-Penetrating Recombinant Peptides for Potential Use in Agricultural Pest Control Applications

    Directory of Open Access Journals (Sweden)

    Eric T. Johnson

    2012-09-01

    Full Text Available Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX from the wolf spider (Lycosa carolinensis. One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.

  20. Selective mono-radioiodination and characterization of a cell-penetrating peptide. L-Tyr-maurocalcine

    Energy Technology Data Exchange (ETDEWEB)

    Ahmadi, Mitra; Bacot, Sandrine; Perret, Pascale; Riou, Laurent; Ghezzi, Catherine [Universite Joseph Fourier, Grenoble (France); INSERM U1039, Grenoble (France). Radiopharmaceutiques Biocliniques; Poillot, Cathy; Cestele, Sandrine [INSERM U836, Grenoble (France). Grenoble Inst. of Neuroscience; Universite Joseph Fourier, Grenoble (France); Desruet, Marie-Dominique [INSERM U1039, Grenoble (France). Radiopharmaceutiques Biocliniques; Couvet, Morgane; Bourgoin, Sandrine; Seve, Michel [CRI-INSERM U823, Grenoble (France). Inst. of Albert Bonniot; Universite Joseph Fourier, Grenoble (France); Waard, Michel de [INSERM U836, Grenoble (France). Grenoble Inst. of Neuroscience; Universite Joseph Fourier, Grenoble (France); Smartox Biotechnologies, Grenoble (France)

    2014-07-01

    Mono-and poly-iodinated peptides form frequently during radioiodination procedures. However, the formation of a single species in its mono-iodinated form is essential for quantitative studies such as determination of tissue concentration or image quantification. Therefore, the aim of the present study was to define the optimal experimental conditions in order to exclusively obtain the mono-iodinated form of L-maurocalcine (L-MCa). L-MCa is an animal venom toxin which was shown to act as a cell-penetrating peptide. In order to apply the current direct radioiodination technique using oxidative agents including chloramine T, Iodo-Gen {sup registered} or lactoperoxidase, an analogue of this peptide containing a tyrosine residue (Tyr-L-MCa) was synthesized and was shown to fold/oxidize properly. The enzymatic approach using lactoperoxidase/H{sub 2}O{sub 2} was found to be the best method for radioiodination of Tyr-L-MCa. MALDI-TOF mass spectrometry analyses were then used for identification of the chromatographic eluting components of the reaction mixtures. We observed that the production of different radioiodinated species depended upon the reaction conditions. Our results successfully described the experimental conditions of peptide radioiodination allowing the exclusive production of the mono-iodinated form with high radiochemical purity and without the need for a purification step. Mono-radioiodination of L-Tyr-MCa will be crucial for future quantitative studies, investigating the mechanism of cell penetration and in vivo biodistribution.

  1. Selective mono-radioiodination and characterization of a cell-penetrating peptide. L-Tyr-maurocalcine

    International Nuclear Information System (INIS)

    Mono-and poly-iodinated peptides form frequently during radioiodination procedures. However, the formation of a single species in its mono-iodinated form is essential for quantitative studies such as determination of tissue concentration or image quantification. Therefore, the aim of the present study was to define the optimal experimental conditions in order to exclusively obtain the mono-iodinated form of L-maurocalcine (L-MCa). L-MCa is an animal venom toxin which was shown to act as a cell-penetrating peptide. In order to apply the current direct radioiodination technique using oxidative agents including chloramine T, Iodo-Gen registered or lactoperoxidase, an analogue of this peptide containing a tyrosine residue (Tyr-L-MCa) was synthesized and was shown to fold/oxidize properly. The enzymatic approach using lactoperoxidase/H2O2 was found to be the best method for radioiodination of Tyr-L-MCa. MALDI-TOF mass spectrometry analyses were then used for identification of the chromatographic eluting components of the reaction mixtures. We observed that the production of different radioiodinated species depended upon the reaction conditions. Our results successfully described the experimental conditions of peptide radioiodination allowing the exclusive production of the mono-iodinated form with high radiochemical purity and without the need for a purification step. Mono-radioiodination of L-Tyr-MCa will be crucial for future quantitative studies, investigating the mechanism of cell penetration and in vivo biodistribution.

  2. Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

    KAUST Repository

    Rydberg, Hanna A.

    2012-07-10

    Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view. © 2012 American Chemical Society.

  3. Cationic cell-penetrating peptides as vehicles for siRNA delivery.

    Science.gov (United States)

    Beloor, Jagadish; Zeller, Skye; Choi, Chang Seon; Lee, Sang-Kyung; Kumar, Priti

    2015-01-01

    RNA interference mediated gene silencing has tremendous applicability in fields ranging from basic biological research to clinical therapy. However, delivery of siRNA across the cell membrane into the cytoplasm, where the RNA silencing machinery is located, is a significant hurdle in most primary cells. Cell-penetrating peptides (CPPs), peptides that possess an intrinsic ability to translocate across cell membranes, have been explored as a means to achieve cellular delivery of siRNA. Approaches using CPPs by themselves or through incorporation into other siRNA delivery platforms have been investigated with the intent of improving cytoplasmic delivery. Here, we review the utilization of CPPs for siRNA delivery with a focus on strategies developed to enhance cellular uptake, endosomal escape and cytoplasmic localization of CPP/siRNA complexes.

  4. Dimerization of a cell-penetrating peptide leads to enhanced cellular uptake and drug delivery

    Directory of Open Access Journals (Sweden)

    Jan Hoyer

    2012-10-01

    Full Text Available Over the past 20 years, cell-penetrating peptides (CPPs have gained tremendous interest due to their ability to deliver a variety of therapeutically active molecules that would otherwise be unable to cross the cellular membrane due to their size or hydrophilicity. Recently, we reported on the identification of a novel CPP, sC18, which is derived from the C-terminus of the 18 kDa cationic antimicrobial protein. Furthermore, we demonstrated successful application of sC18 for the delivery of functionalized cyclopentadienyl manganese tricarbonyl (cymantrene complexes to tumor cell lines, inducing high cellular toxicity. In order to increase the potential of the organometallic complexes to kill tumor cells, we were looking for a way to enhance cellular uptake. Therefore, we designed a branched dimeric variant of sC18, (sC182, which was shown to have a dramatically improved capacity to internalize into various cell lines, even primary cells, using flow cytometry and fluorescence microscopy. Cell viability assays indicated increased cytotoxicity of the dimer presumably caused by membrane leakage; however, this effect turned out to be dependent on the specific cell type. Finally, we could show that conjugation of a functionalized cymantrene with (sC182 leads to significant reduction of its IC50 value in tumor cells compared to the respective sC18 conjugate, proving that dimerization is a useful method to increase the drug-delivery potential of a cell-penetrating peptide.

  5. Metabolic cleavage of cell-penetrating peptides in contact with epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg;

    2004-01-01

    We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) and...... penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting...... models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C...

  6. Poly(NIPAm-AMPS) nanoparticles for targeted delivery of anti-inflammatory cell penetrating peptides

    Science.gov (United States)

    Bartlett, Rush Lloyd, II

    Inflammatory diseases such as osteoarthritis and rheumatoid arthritis cause $127.8 billion in US healthcare expenditures each year and are the cause of disability for 27% of disabled persons in the United States. Current treatment options rarely halt disease progression and often result in significant unwanted and debilitating side effects. Our laboratory has previously developed a family of cell penetrating peptides (CPPs) which inhibit the activity of mitogen activated protein kinase activate protein kinase 2 (MK2). MK2 mediates the inflammatory response by activating Tristetraprline (TTP). Once activated, TTP rapidly stabilizes AU rich regions of pro-inflammatory cytokine mRNA which allows translation of pro-inflammatory cytokines to occur. Blocking MK2 with our labs CPPs yields a decrease in inflammatory activity but CPPs by are highly non specific and prone to rapid enzymatic degradation in vivo.. In order to increase the potency of MK2 inhibiting CPPs we have developed a novel nanoparticle drug carrier composed of poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid). This drug carrier has been shown to have preliminary efficacy in vitro and ex vivo for suppressing pro-inflammatory cytokine production when releasing CPPs. This thesis will present progress made on three aims: Specific Aim 1) Create and validate a NIPAm based drug delivery system that mimics the binding and release previously observed between cell penetrating peptides and glycosaminoglycans. Specific Aim 2) Engineer degradability into poly(NIPAm-AMPS) nanoparticles to enable more drug to be released and qualify that system in vitro. Specific Aim 3) Validate poly(NIPAm-AMPS) nanoparticles for targeted drug delivery in an ex vivo inflammatory model. Overall we have developed a novel anionic nanoparticle system that is biocompatible and efficient at loading and releasing cell penetrating peptides to inflamed tissue. Once loaded with a CPP the nanoparticle drug complex is

  7. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

    DEFF Research Database (Denmark)

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter E;

    2015-01-01

    hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its......Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers...

  8. Distinct behaviour of the homeodomain derived cell penetrating peptide penetratin in interaction with different phospholipids.

    Directory of Open Access Journals (Sweden)

    Ofelia Maniti

    Full Text Available BACKGROUND: Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC and phosphatidylethanolamine (PE to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β-sheet contributions. CONCLUSIONS/SIGNIFICANCE: These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution.

  9. Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

    Directory of Open Access Journals (Sweden)

    Sofie Stalmans

    Full Text Available Cell-penetrating peptides (CPPs are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB. However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10 and TP10-2, were determined. The results of the multiple time regression (MTR analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min, 5.63 μl/(g × min and 6.02 μl/(g × min, respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

  10. Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

    Science.gov (United States)

    Stalmans, Sofie; Bracke, Nathalie; Wynendaele, Evelien; Gevaert, Bert; Peremans, Kathelijne; Burvenich, Christian; Polis, Ingeborgh; De Spiegeleer, Bart

    2015-01-01

    Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min), 5.63 μl/(g × min) and 6.02 μl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

  11. Cell penetrating peptide delivery of splice directing oligonucleotides as a treatment for Duchenne muscular dystrophy.

    Science.gov (United States)

    Betts, Corinne A; Wood, Matthew J A

    2013-01-01

    Duchenne muscular dystrophy is a severe, X-linked muscle wasting disorder caused by the absence of an integral structural protein called dystrophin. This is caused by mutations or deletions in the dystrophin gene which disrupt the reading frame, thereby halting the production of a functional protein. A number of potential therapies have been investigated for the treatment of this disease including utrophin upregulation, 'stop-codon read through' aminoglycosides and adeno-associated virus gene replacement as well as stem cell therapy. However, the most promising treatment to date is the use of antisense oligonucleotides which cause exon skipping by binding to a specific mRNA sequence, skipping the desired exon, thereby restoring the reading frame and producing a truncated yet functional protein. The results from recent 2'OMePS and morpholino clinical trials have renewed hope for Duchenne patients; however in vivo studies in a mouse model, mdx, have revealed low systemic distribution and poor delivery of oligonucleotides to affected tissues such as the brain and heart. However a variety of cell penetrating peptides directly conjugated to antisense oligonucleotides have been shown to enhance delivery in Duchenne model systems with improved systemic distribution and greater efficacy compared to 'naked' antisense oligonucleotides. These cell penetrating peptides, combined with an optimised dose and dosing regimen, as well as thorough toxicity profile have the potential to be developed into a promising treatment which may be progressed to clinical trial. PMID:23140454

  12. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  13. Enhancing tumor-specific intracellular delivering efficiency of cell-penetrating peptide by fusion with a peptide targeting to EGFR.

    Science.gov (United States)

    Nguyen, Long The; Yang, Xu-Zhong; Du, Xuan; Wang, Jia-Wei; Zhang, Rui; Zhao, Jian; Wang, Fu-Jun; Dong, Yang; Li, Peng-Fei

    2015-05-01

    Cell-penetrating peptides (CPPs) are well known as intracellular delivery vectors. However, unsatisfactory delivery efficiency and poor specificity are challenging barriers to CPP applications at the clinical trial stage. Here, we showed that S3, an EGFR-binding domain derived from vaccinia virus growth factor, when fused to a CPP such as HBD or TAT can substantially enhance its internalization efficiency and tumor selectivity. The uptake of S3-HBD (S3H) recombinant molecule by tumor cells was nearly 80 folds increased compared to HBD alone. By contrast, the uptake of S3H by non-neoplastic cells still remained at a low level. The specific recognition between S3 and its receptor, EGFR, as well as between HBD and heparan sulfate proteoglycans on the cell surface was essential for these improvements, suggesting a syngeneic effect between the two functional domains in conjugation. This syngeneic effect is likely similar to that of the heparin-binding epidermal growth factor, which is highly abundant particularly in metastatic tumors. The process that S3H entered cells was dependent on time, dosage, and energy, via macropinocytosis pathway. With excellent cell-penetrating efficacy and a novel tumor-targeting ability, S3H appears as a promising candidate vector for targeted anti-cancer drug delivery. PMID:25655386

  14. Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

    Science.gov (United States)

    Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

    2016-01-01

    Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes. PMID:27516189

  15. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides.

    Science.gov (United States)

    Agrawal, Piyush; Bhalla, Sherry; Usmani, Salman Sadullah; Singh, Sandeep; Chaudhary, Kumardeep; Raghava, Gajendra P S; Gautam, Ankur

    2016-01-01

    CPPsite 2.0 (http://crdd.osdd.net/raghava/cppsite/) is an updated version of manually curated database (CPPsite) of cell-penetrating peptides (CPPs). The current version holds around 1850 peptide entries, which is nearly two times than the entries in the previous version. The updated data were curated from research papers and patents published in last three years. It was observed that most of the CPPs discovered/ tested, in last three years, have diverse chemical modifications (e.g. non-natural residues, linkers, lipid moieties, etc.). We have compiled this information on chemical modifications systematically in the updated version of the database. In order to understand the structure-function relationship of these peptides, we predicted tertiary structure of CPPs, possessing both modified and natural residues, using state-of-the-art techniques. CPPsite 2.0 also maintains information about model systems (in vitro/in vivo) used for CPP evaluation and different type of cargoes (e.g. nucleic acid, protein, nanoparticles, etc.) delivered by these peptides. In order to assist a wide range of users, we developed a user-friendly responsive website, with various tools, suitable for smartphone, tablet and desktop users. In conclusion, CPPsite 2.0 provides significant improvements over the previous version in terms of data content. PMID:26586798

  16. Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides

    Directory of Open Access Journals (Sweden)

    Aubin eMoutal

    2015-01-01

    Full Text Available The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2 is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3 conjugated to the HIV transactivator of transcription (TAT protein’s cationic cell penetrating peptide motif (CPP protected neurons in the face of toxic levels of Ca2+ influx leaked in via N-methyl-D-aspartate receptor (NMDAR hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9, hydrophobic (membrane transport sequence (MTS of k-fibroblast growth factor or amphipathic (model amphipathic peptide (MAP CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA-evoked Ca2+ influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca2+ influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 minutes, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (> 24 hours treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

  17. Comparative mechanisms of protein transduction mediated by cell-penetrating peptides in prokaryotes.

    Science.gov (United States)

    Liu, Betty Revon; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2015-04-01

    Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

  18. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection

    Science.gov (United States)

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-04-01

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL-1. Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid

  19. Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine.

    Directory of Open Access Journals (Sweden)

    Divyamani Srinivasan

    Full Text Available BACKGROUND: Cell-penetrating peptides (CPPs can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6-carboxytetramethylrhodamine (TMR. METHODOLOGY/PRINCIPAL FINDINGS: We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers. CONCLUSIONS/SIGNIFICANCE: Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.

  20. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    Science.gov (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  1. Nanocarriers Conjugated with Cell Penetrating Peptides: New Trojan Horses by Modern Ulysses.

    Science.gov (United States)

    Zappavigna, Silvia; Misso, Gabriella; Falanga, Annarita; Perillo, Emiliana; Novellino, Ettore; Galdiero, Massimiliano; Grieco, Paolo; Caraglia, Michele; Galdiero, Stefania

    2016-01-01

    Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover, recent literature has illustrated the use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanced drug delivery. CPPs exploit their ability to enter cells and enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctive properties of nanocarriers (NC) based systems provide unforeseen benefits over pure drugs for biomedical applications and constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have to be overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use both for effective tumor therapy and gene therapy. A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs' uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amount will escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, we will discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems. PMID:27087493

  2. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment.

    Science.gov (United States)

    Yamashita, Hiroko; Kato, Takuma; Oba, Makoto; Misawa, Takashi; Hattori, Takayuki; Ohoka, Nobumichi; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2016-01-01

    Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1-5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-Pro(NH2))3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-Pro(Gu))3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-Pro(Gu))2-(l-Arg)4-l-Pro(Gu)-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-Pro(Gu))3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-Pro(NH2) and l-Pro(Gu)), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-Pro(Gu) exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-Pro(Gu)-containing peptide 3 may be a useful candidate as a gene delivery carrier. PMID:27609319

  3. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment

    Science.gov (United States)

    Yamashita, Hiroko; Kato, Takuma; Oba, Makoto; Misawa, Takashi; Hattori, Takayuki; Ohoka, Nobumichi; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2016-01-01

    Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1–5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier. PMID:27609319

  4. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides.

    Science.gov (United States)

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale

    2014-02-01

    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  5. Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient.

    Science.gov (United States)

    Madani, Fatemeh; Abdo, Rania; Lindberg, Staffan; Hirose, Hisaaki; Futaki, Shiroh; Langel, Ulo; Gräslund, Astrid

    2013-04-01

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  6. Cellular uptake but low permeation of human calcitonin-derived cell penetrating peptides and Tat(47-57) through well-differentiated epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Krauss, Ulrike; Beck-Sickinger, Annette G;

    2004-01-01

    To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers.......To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers....

  7. Cell-penetrating TAT peptide in drug delivery systems: Proteolytic stability requirements

    Science.gov (United States)

    Koren, Erez; Apte, Anjali; Sawant, Rupa R.; Grunwald, Jacob; Torchilin, Vladimir P.

    2012-01-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a ‘short’ PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function. PMID:21438724

  8. Enhanced cellular delivery of cell-penetrating peptide-peptide nucleic acid conjugates by photochemical internalization

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    )) or tetraphenylporphyrin tetrasulfonic acid (TPPS). Cellular uptake of the PNA conjugates were evaluated by using a sensitive cellular method with HeLa pLuc705 cells based on the splicing correction of luciferase gene by targeting antisense oligonucleotides to a cryptic splice site of the mutated luciferase gene....... The cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA....

  9. Conjugation of a cell-penetrating peptide to parathyroid hormone affects its structure, potency, and transepithelial permeation

    DEFF Research Database (Denmark)

    Kristensen, Mie; de Groot, Anne Marit; Berthelsen, Jens;

    2015-01-01

    hormone, i.e. PTH(1-34), and to evaluate the effect with regards to secondary structure, potency in Saos-2 cells, immunogenicity, safety as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative......Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid...

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

    Directory of Open Access Journals (Sweden)

    Taku Kaitsuka

    2015-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  12. Hierarchy of Specific Lipid-Peptide Interactions Produces the Activity of Cell-penetrating and Cell-permeating Peptides

    Science.gov (United States)

    Davis, Matthew; Parente, Daniel; Gordon, Vernita; Mishra, Abhijit; Schmidt, Nathan; Yang, Lihua; Coridan, Robert; Som, Abhigyan; Tew, Gregory; Wong, Gerard

    2008-03-01

    Protein transduction domains can cross cell membranes with high efficiency, even when carrying a variety of cargos, and thus has strong biotechnological potential. The molecular mechanism of entry, however, is not well understood. We use small-angle x-ray scattering (SAXS) and confocal microscopy to systematically study the interaction of the TAT and ANTP PTD with model membranes of variable composition. Their membrane transduction activity requires the presence of both PE and PS lipids in the membrane. Antimicrobial peptides (AMP's) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogs of AMP's, such as the family of phenylene ethynylene antimicrobial oligomers (AMO's), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. PE lipid greatly enhances permeating activity of AMO in these membranes, showing the importance of specific lipid composition for the activity of cell-permeating peptides. Since bacterial cell membranes are richer in PE lipids than are eukaryotic cell membranes, this may indicate a mechanism for antimicrobial specificity.

  13. Design and screening of a glial cell-specific, cell penetrating peptide for therapeutic applications in multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Corey Heffernan

    Full Text Available Multiple Sclerosis (MS is an autoimmune, neurodegenerative disease of the central nervous system (CNS characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i modulation of the host immune system; and/or (ii transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types, and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i human neural cells and (ii human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP to mature oligodendrocytes 3-6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS.

  14. Stearylated antimicrobial peptide [D]-K6L9 with cell penetrating property for efficient gene transfer.

    Science.gov (United States)

    Zhang, Wei; Song, Jingjing; Liang, Ranran; Zheng, Xin; Chen, Jianbo; Li, Guolin; Zhang, Bangzhi; Wang, Kairong; Yan, Xiang; Wang, Rui

    2013-08-01

    Stearyl-cell penetrating peptides (CPPs) have been proved to be efficient nonviral gene vectors. Due to the similarities between antimicrobial peptides and CPPs, we constructed a novel type of gene vectors by introducing stearyl moiety to the N-terminus of antimicrobial peptide [D]-K6L9. In this study, stearyl-[D]-K6L9 delivered plasmids into cells by clathrin- and caveolin-mediated endocytosis. Gratifyingly, stearyl-[D]-K6L9 exhibited high transfection efficiency and almost reached the level of Lipofectamine 2000. Taken together, the combination of the stearyl moiety with [D]-K6L9 provides a novel framework for the development of excellent nonviral gene vectors. PMID:23727033

  15. Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides.

    Science.gov (United States)

    Keller, Andrea-Anneliese; Breitling, Reinhard; Hemmerich, Peter; Kappe, Katarina; Braun, Maria; Wittig, Berith; Schaefer, Buerk; Lorkowski, Stefan; Reissmann, Siegmund

    2014-02-01

    Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules), and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al., ]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis.

  16. Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

    Science.gov (United States)

    Oehlke, Johannes; Wallukat, Gerd; Wolf, Yvonne; Ehrlich, Angelika; Wiesner, Burkhard; Berger, Hartmut; Bienert, Michael

    2004-07-01

    In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.

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

    International Nuclear Information System (INIS)

    Highlights: ► Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. ► Dimer formation enhances peptiplex stability, resulting in increased transfection. ► 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 its stronger binding to DNA.

  18. Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers.

    Science.gov (United States)

    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.

  19. Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles.

    Science.gov (United States)

    Sharmin, Sabrina; Islam, Md Zahidul; Karal, Mohammad Abu Sayem; Alam Shibly, Sayed Ul; Dohra, Hideo; Yamazaki, Masahito

    2016-08-01

    The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary

  20. riDOM, a cell-penetrating peptide. Interaction with DNA and heparan sulfate.

    Science.gov (United States)

    Québatte, Gabriela; Kitas, Eric; Seelig, Joachim

    2013-09-19

    DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin.

  1. 细胞穿透肽的研究进展%Recent advances in research on cell penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    李凤英; 何京; 钟海军

    2013-01-01

    细胞穿透肽(cell penetrating peptides,CPP)是一类能够通过生物膜进入细胞的短肽,它具有一些其他运载系统无法比拟的优点:低浓度条件下,可以穿过细胞膜进入细胞并且不会对膜造成明显破坏和损伤;能介导各种物质包括小分子、核酸、蛋白多肽以及纳米粒子等入胞;高效、低毒.本文就CPP的分类、与载物的连接方式、穿膜机制、应用和常用研究方法等方面进行系统的综述,并对CPP的临床应用前景进行展望.%Cell penetrating peptides (CPP) are a class of peptides of less than 35 amino acids with a remarkable capacity for membrane transportation.Compared with other delivery systems,the CPP-based system has several advantages.These peptides are able to penetrate into cells at low micromolar concentrations in vitro and in vivo without causing significant membrane damage.Furthermore,they constitute very promising tools for cellular import of various substances including small molecules,nucleic acids,proteins and nanoparticles.In this review,the categories,chemical linkage with cargoes,cellular uptake mechanism,applications and research methods of CPP are systematically summarized.The research future of CPP,especially the future of its clinical application,is also introduced.

  2. Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

    Science.gov (United States)

    Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

    2011-11-01

    Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

  3. Cell-penetration by Co(III)cyclen-based peptide-cleaving catalysts selective for pathogenic proteins of amyloidoses.

    Science.gov (United States)

    Chei, Woo Suk; Lee, Joo-Won; Kim, Jae Bum; Suh, Junghun

    2010-07-15

    Derivatives of the Co(III) complex of 1,4,7,10-tetraazacyclododecane (cyclen) with various organic pendants have been reported as target-selective peptide-cleaving catalysts, which can be exploited as catalytic drugs. In order to provide a firm basis for the catalytic drugs based on Co(III)cyclen, the ability of the Co(III)cyclen-containing peptide-cleaving catalysts to penetrate animal cells such as mouse fibroblast NIH-3T 3 or human embryonic kidney (HEK) 293 cells is demonstrated in the present study. Since the catalysts destroy pathogenic proteins for amyloidoses, results of the present study are expected to initiate extensive efforts to obtain therapeutically safe catalytic drugs for amyloidoses such as Alzheimer's disease, type 2 diabetes mellitus, Parkinson's disease, Huntington's disease, mad cow disease, and so on. PMID:20542701

  4. Spotlight on Human LL-37, an Immunomodulatory Peptide with Promising Cell-Penetrating Properties

    Directory of Open Access Journals (Sweden)

    Marc Ferdinand Lensink

    2010-11-01

    Full Text Available Cationic antimicrobial peptides are major components of innate immunity and help control the initial steps of the infectious process. They are expressed not only by immunocytes, but also by epithelial cells. They share an amphipathic secondary structure with a polar cationic site, which explains their tropism for prokaryote membranes and their hydrophobic site contributing to the destructuration of these membranes. LL-37 is the only cationic antimicrobial peptide derived from human cathelicidin. LL-37 can also cross the plasma membrane of eukaryotic cells, probably through special domains of this membrane called lipid rafts. This transfer could be beneficial in the context of vaccination: the activation of intracellular toll-like receptors by a complex formed between CpG oligonucleotides and LL-37 could conceivably play a major role in the building of a cellular immunity involving NK cells.

  5. Combined effect of a peptide–morpholino oligonucleotide conjugate and a cell-penetrating peptide as an antibiotic

    OpenAIRE

    Wesolowski, Donna; Alonso, Dulce; Altman, Sidney

    2013-01-01

    A cell-penetrating peptide (CPP)–morpholino oligonucleotide (MO) conjugate (PMO) that has an antibiotic effect in culture had some contaminating CPPs in earlier preparations. The mixed conjugate had gene-specific and gene-nonspecific effects. An improved purification procedure separates the PMO from the free CPP and MO. The gene-specific effects are a result of the PMO, and the nonspecific effects are a result of the unlinked, unreacted CPP. The PMO and the CPP can be mixed together, as has b...

  6. The spacer arm length in cell-penetrating peptides influences chitosan/siRNA nanoparticle delivery for pulmonary inflammation treatment

    Science.gov (United States)

    Jeong, Eun Ju; Choi, Moonhwan; Lee, Jangwook; Rhim, Taiyoun; Lee, Kuen Yong

    2015-11-01

    Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of R9Gn-chitosan/siRNA nanoparticles were investigated in vitro. Increasing the spacing arm length did not significantly affect the complex formation between R9Gn-chitosan and siRNA. However, R9G10-chitosan was much more effective in delivering genes both in vitro and in vivo compared with non-modified chitosan (without the peptide) and R9-chitosan (without the spacer arm). Chitosan derivatives modified with oligoarginine containing a spacer arm can be considered as potential delivery vehicles for various genes.Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of

  7. Cell-penetrating peptide-doxorubicin conjugate loaded NGR-modified nanobubbles for ultrasound triggered drug delivery.

    Science.gov (United States)

    Lin, Wen; Xie, Xiangyang; Deng, Jianping; Liu, Hui; Chen, Ying; Fu, Xudong; Liu, Hong; Yang, Yang

    2016-01-01

    A new drug-targeting system for CD13(+) tumors has been developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, the CPP-doxorubicin conjugate (CPP-DOX) was entrapped in the asparagine-glycine-arginine (NGR) peptide modified NB (CPP-DOX/NGR-NB) and the penetration of CPP-DOX was temporally masked; local ultrasound stimulation could trigger the CPP-DOX release from NB and activate its penetration. The CPP-DOX/NGR-NBs had particle sizes of about 200 nm and drug entrapment efficiency larger than 90%. In vitro release results showed that over 85% of the encapsulated DOX or CPP-DOX would release from NBs in the presence of ultrasound, while less than 1.5% of that (30 min) without ultrasound. Cell experiments showed the higher cellular CPP-DOX uptake of CPP-DOX/NGR-NB among the various NB formulations in Human fibrosarcoma cells (HT-1080, CD13(+)). The CPP-DOX/NGR-NB with ultrasound treatment exhibited an increased cytotoxic activity than the one without ultrasound. In nude mice xenograft of HT-1080 cells, CPP-DOX/NGR-NB with ultrasound showed a higher tumor inhibition effect (3.1% of T/C%, day 24), longer median survival time (50 days) and excellent body safety compared with the normal DOX injection group. These results indicate that the constructed vesicle would be a promising drug delivery system for specific cancer treatment.

  8. Fusion of cell-penetrating peptides to thermally responsive biopolymer improves tumor accumulation of p21 peptide in a mouse model of pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Walker LR

    2014-10-01

    Full Text Available Leslie R Walker,1 Jung Su Ryu,1 Eddie Perkins,2 Lacey R McNally,3 Drazen Raucher1 1Department of Biochemistry, 2Department of Neurosurgery, University of Mississippi Medical Center, Jackson, MS, USA; 3Division of Hematology and Oncology, University of Louisville, Louisville, KY, USAAbstract: Current therapies for the treatment of pancreatic cancer are limited. The limitations of this type of treatment are abundant. The majority of chemotherapeutic agents used in clinics are highly toxic to both tumor cells and normal tissues due to the lack of specificity. Resistance can develop due to overexposure of these agents. To address these issues, these agents must be made more exclusive toward the tumor site. We have developed a macromolecular carrier based on the sequence of the biopolymer elastin-like polypeptide (ELP that is able to aggregate upon reaching the externally heated tumor environment. This carrier is specific to the tumor as it only aggregates at the heated tumor site. ELP is soluble below its transition temperature but will aggregate when the temperature is raised above its transition temperature. ELP was modified by p21, a cell cycle inhibitory peptide, and the addition of Bac, a cell-penetrating peptide with nuclear localization capabilities. In this study, p21-ELP-Bac and its control, ELP-p21, were used in cell proliferation studies using the pancreatic cancer cell lines Panc-1, MiaPaca-2, and S2013. ELP-p21 had little effect on proliferation, while the half maximal inhibitory concentration of p21-ELP-Bac was ~30 µM. As translocation across the plasma membrane is a limiting step for delivery of macromolecules, these polypeptides were utilized in a pancreatic xenograft model to study the plasma clearance, biodistribution, tumor accumulation, and tumor reduction capabilities of the polypeptide with and without a cell-penetrating peptide.Keywords: elastin-like polypeptide, peptide, targeted drug delivery, macromolecule

  9. State of the Art in the Studies on Crotamine, a Cell Penetrating Peptide from South American Rattlesnake

    Directory of Open Access Journals (Sweden)

    Irina Kerkis

    2014-01-01

    Full Text Available Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP with a more pronounced antifungal activity. In contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. In vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. The structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.

  10. Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides.

    Science.gov (United States)

    Bartlett, Rush L; Medow, Matthew R; Panitch, Alyssa; Seal, Brandon

    2012-04-01

    Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.

  11. Effective Penetration of Cell-permeable Peptide Mimic of Tyrosine Residue 654 Domain of β-catenin into Human Renal Tubular Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    ZENG Rui; XU Gang; HAN Min; LIU Wei; LIU Xiaocheng

    2007-01-01

    Phosphorylation of β-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition (EMT). Introducing mimic peptide of tyrosine residue 654 domain of β-catenin into cells may influence phosphorylation of β-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell perme-able peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic pep-tide of β-catenin (PM) with fused penetratin was constructed, purified and then detected for the pene-tration of the mimic peptide into human renal tubular epithelial cells (HK-2). The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of β-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.

  12. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

    Science.gov (United States)

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

    2014-12-01

    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  13. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems☆

    Science.gov (United States)

    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

  14. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems

    Directory of Open Access Journals (Sweden)

    Mariana H. Massaoka

    2014-01-01

    Full Text Available 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.

  15. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    Science.gov (United States)

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

  16. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    Science.gov (United States)

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. PMID:25797209

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

    Science.gov (United States)

    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.

  18. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Directory of Open Access Journals (Sweden)

    Suh JS

    2014-03-01

    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

  19. PREPARATION OF CHEMICAL AND PHYSICAL CONJUGATES OF SELF-ASSEMBLING NANOPARTICLES WITH CELL-PENETRATING PEPTIDE AND DOXORUBICIN

    Directory of Open Access Journals (Sweden)

    Zhadyra Sagykyzy Shagyrova

    2015-09-01

    Full Text Available Abstract: Nano-sized carriers can help to reduce toxicity and improve clinical efficacy of drugs. Virus-like particles (VLPs are biocompatible and biodegradable self-assembling nanoparticles, which show great promise as carriers for substances for targeted delivery and controlled release. Either chemical conjugation of physical incorporation without formation of covalent bonds is possible to load substances of interest into VLPs.Objectives: To produce VLPs from recombinant viral capsid protein (HBcAg and test feasibility of methods of formation of chemical and physical conjugates of VLPs with substances of pharmacological interest.Methods: Virus-like particles composed from recombinant hepatitis B core antigen (HBcAg were produced by recombinant expression in E.coli and purified by successive centrifugation through sucrose gradients. Peptide transportan 10 was synthesized and used for carbodiimide (EDC-mediated conjugation to VLPs. Doxorubicin (DOX was loaded into the nucleic acid-containing VLPs to form physical conjugate.Results: VLPs with chemically attached moieties of cell-penetrating peptide transportan 10 were produced. The conjugate was examined in SDS-PAGE to confirm presence of conjugation products. Conjugation efficiency (molar ration peptide/protein in the conjugate reaches 0.5:1 (i.e. 50% of protein chains have one attached peptide moiety. The nucleic acid-containing VLPs can be loaded with the DOX forming stable non-covalent physical conjugate.Conclusion: Recombinantly expressed VLPs allow easy attaching of small molecules making them a convenient platform to develop drug carriers.

  20. A novel cell penetrating peptide carrier for the delivery of nematocidal proteins drug

    Science.gov (United States)

    Kim, Jea Hyun

    Nematodes have recently become a primary source of harmful diseases to the environment that inflict harsh damages to pine trees and marine species. However, nematodes cannot be killed by normal pesticides or chemicals due to their thick outer protective layer mainly composed of collagen and cuticles. Thus, a novel approach to trigger intracellular delivery of chemicals through the layers of nematodes is required. In this study, the selection of the novel CPP was carefully progressed through protein database and serial digested fragmentation, internalization of each amino sequence was analyzed through flow cytometry and confocal microscope. As one of the most effective CPP material, JH 1.6 was compared with other major CPPs and its cellular toxicity was investigated. Furthermore, JH 1.6 was attached to various RNA, DNA, and proteins and internalization efficiency was evaluated for mammalian cells. To examine its effects on nematodes in vivo, JH 1.6 was conjugated with nematocidal protein - botulinum neurotoxin (BnT) and treated in C.elegans as a model animal. The results showed that JH 1.6 had high relative internalization rate and low cellular toxicity compared to other major CPP such as TAT and GV1001 peptides.

  1. Cell-penetrating peptide derived from human eosinophil cationic protein inhibits mite allergen Der p 2 induced inflammasome activation.

    Directory of Open Access Journals (Sweden)

    Sheng-Jie Yu

    Full Text Available Newly discovered cell penetration peptides derived from human eosinophil cationic proteins (CPPecp have the characteristic of cell internalization, but the effect of CPPecp on immunomodulation has not been clarified. House dust mite (HDM major allergen, Der p 2, can induce proinflammatory cytokine production which contributes to airway inflammation and allergic asthma. However, the mechanism of Der p 2 on NLRP3 inflammasome activation remains unclear. The aim of this study was to investigate the immunomodulatory effect of CPPecp on inhibition of Der p 2 induced inflammasome activation. We showed that proinflammatory cytokines IL-1β, IL-6 and IL-8 were significantly upregulated in peripheral blood mononuclear cells (PBMCs derived from HDM allergic patients after Der p 2 stimulation. Expression of NLRP3, ASC, Caspase-1, IL-1β and Caspase-1 activity was upregulated in THP-1 cells after Der p 2 stimulation. Proinflammatory cytokine production, NLRP3 inflammasome activation and caspase-1 activity were downregulated in THP-1 cells and CD14+ cells co-cultured with Der p 2 and CPPecp. The immunomodulatory effect of CPPecp was through upregulation of IFN-α production but not induction of autophagy. These results suggested Der p 2 plays an important role in NLRP3 inflammasome activation and CPPecp has the potential to be a novel anti-inflammatory agent for allergic inflammation treatment in the future.

  2. Single-cell resolution imaging of retinal ganglion cell apoptosis in vivo using a cell-penetrating caspase-activatable peptide probe.

    Directory of Open Access Journals (Sweden)

    Xudong Qiu

    Full Text Available Peptide probes for imaging retinal ganglion cell (RGC apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in

  3. Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state.

    Directory of Open Access Journals (Sweden)

    Susanne Fanghänel

    Full Text Available Structure analysis of the cell-penetrating peptide transportan 10 (TP10 revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan was found to exhibit prominent characteristics of (i amphiphilic α-helices, (ii intrinsically disordered peptides, as well as (iii β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg as a reporter group for (19F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the

  4. Conjugation of doxorubicin to cell penetrating peptides sensitizes human breast MDA-MB 231 cancer cells to endogenous TRAIL-induced apoptosis.

    OpenAIRE

    Aroui, Sonia; Brahim, Souhir; Hamelin, Jocelyne; De Waard, Michel; Bréard, Jacqueline; Kenani, Abderraouf

    2009-01-01

    International audience Previous work from our laboratory has shown that coupling doxorubicin (Dox) to cell penetrating peptides (Dox-CPPs) is a good strategy to overcome Dox resistance in MDA-MB 231 breast cancer cells. We also reported that, in contrast to unconjugated Dox-induced cell death, the increase in apoptotic response does not involve the mitochondrial apoptotic pathway. In this study, we demonstrate that both Dox and Dox-CPPs can increase the density of the TRAIL receptors DR4 a...

  5. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    OpenAIRE

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A; Williams, Donna L.; Deuss, Peter; Gait, Michael J.

    2015-01-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO ...

  6. Research Progress of Cell -penetrating Peptides Applied in Vaccine%穿膜肽在疫苗中应用的研究进展

    Institute of Scientific and Technical Information of China (English)

    范兴琼; 吴双林; 刘成倩; 易建中

    2012-01-01

    Since the discovery of the function of protein transduction, cell- penetrating peptides (CPP) provides a novel approach for gene therapy and vaccine development. According to recent studies on the CPP species, structure, membrane -penetrating mechanisms and relative applications, cell -penetrating peptides applied in the DNA vaccine and tumor vaccine were reviewed and prospected.%穿膜肽自发现以来,因其独特的蛋白转导功能,为基因治疗和新型疫苗的开发等开辟了新的途径。通过综述穿膜肽的种类、结构特点、作用机制及其在疫苗中的应用,为进一步研发DNA疫苗和肿瘤疫苗奠定基础。

  7. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania.

    Science.gov (United States)

    Luque-Ortega, Juan Román; van't Hof, Wim; Veerman, Enno C I; Saugar, José M; Rivas, Luis

    2008-06-01

    Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed. PMID:18230684

  8. Synergistic Enhancement of Antitumor Efficacy by PEGylated Multi-walled Carbon Nanotubes Modified with Cell-Penetrating Peptide TAT

    Science.gov (United States)

    Hu, Shanshan; Wang, Tong; Pei, Xibo; Cai, He; Chen, Junyu; Zhang, Xin; Wan, Qianbing; Wang, Jian

    2016-10-01

    In the present study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to PEGylated multi-walled carbon nanotubes (MWCNTs) to develop a highly effective antitumor drug delivery system. FITC was conjugated on MWCNTs-polyethylene glycol (PEG) and MWCNTs-PEG-TAT to provide fluorescence signal for tracing the cellular uptake of the nanocarrier. After loaded with an anticancer agent, doxorubicin (DOX) via π - π stacking interaction, the physicochemical characteristics, release profile and biological evaluation of the obtained nano-sized drug carrier were investigated. The DOX loaded MWCNTs-PEG and MWCNTs-PEG-TAT drug carriers both displayed appropriate particle size, excellent stability, high drug loading, and pH-dependent drug release profile. Nevertheless, compared with DOX-MWCNTs-PEG, DOX-MWCNTs-PEG-TAT showed improved cell internalization, intracellular distribution and potentiated anticancer efficacy due to the TAT-mediated membrane translocation, endosomal escape and nuclear targeting. Furthermore, the therapeutic efficacy of DOX was not compromised after being conjugated with MWCNTs-PEG-TAT and the proposed nanocarrier was also confirmed to have a good biocompatibility. In conclusion, our results suggested that the unique combination of TAT and MWCNTs as a multifunctional drug delivery system might be a powerful tool for improved anticancer drug development.

  9. Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent,diethylenetriamine pentaacetic acid gadolinium

    Institute of Scientific and Technical Information of China (English)

    GUO You-min; LIU Min; YANG Jun-le; GUO Xiao-juan; WANG Si-cen; DUAN Xiao-yi; WANG Peng

    2007-01-01

    Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T1WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide reduction assay (MTT). Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging (T1WI) signal, and that the T1WI signal intensity was increasing in a time-dependent manner (r=0.972, P=0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P=0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F=0.006, P=1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC

  10. Antitumor activity of tripterine via cell-penetrating peptide-coated nanostructured lipid carriers in a prostate cancer model

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    Yuan L

    2013-11-01

    Full Text Available Ling Yuan,1 Congyan Liu,2 Yan Chen,2 Zhenhai Zhang,2 Lei Zhou,1 Ding Qu2 1Department of Pharmaceutics, School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 2Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu, People's Republic of China Background: The purpose of this study was to evaluate the antitumor effect of cell-penetrating peptide-coated tripterine-loaded nanostructured lipid carriers (CT-NLC on prostate tumor cells in vitro and in vivo. Methods: CT-NLC were developed to improve the hydrophilicity of tripterine. The antiproliferative effects of CT-NLC, tripterine-loaded nanostructured lipid carriers (T-NLC, and free tripterine in a human prostatic carcinoma cell line (PC-3 and a mouse prostate carcinoma cell line (RM-1 were evaluated using an MTT assay. The advantage of CT-NLC over T-NLC and free tripterine with regard to antitumor activity in vivo was evaluated in a prostate tumor-bearing mouse model. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content was investigated by enzyme-linked immunosorbent assay to determine the effect of CT-NLC, T-NLC, and free tripterine on immune responses. Histologic and TUNEL assays were carried out to investigate the mechanisms of tumor necrosis and apoptosis. Results: CT-NLC, T-NLC, and free tripterine showed high antiproliferative activity in a dose-dependent manner, with an IC50 of 0.60, 0.81, and 1.02 µg/mL in the PC-3 cell line and 0.41, 0.54, and 0.89 µg/mL in the RM-1 cell line after 36 hours. In vivo, the tumor inhibition rates for cyclophosphamide, high-dose (4 mg/kg and low-dose (2 mg/kg tripterine, high-dose (4 mg/kg and low-dose (2 mg/kg T-NLC, high-dose (4 mg/kg and low-dose (2 mg/kg CT-NLC were 76.51%, 37.07%, 29.53%, 63.56%, 48.25%, 72.68%, and 54.50%, respectively, showing a dose-dependent pattern. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content

  11. Curb Challenges of the “Trojan Horse” Approach: Smart Strategies in Achieving Effective yet Safe Cell-penetrating Peptide-based Drug Delivery

    OpenAIRE

    Huang, Yongzhuo; Jiang, Yifan; Wang, Huiyuan; Wang, Jianxin; Shin, Meong Cheol; Byun, Youngro; He, Huining; Liang, Yanqin; Yang, Victor C.

    2013-01-01

    Cell-penetrating peptide (CPP)-mediated intracellular drug delivery system, often specifically termed as “the Trojan horse approach”, has become the “holy grail” in achieving effective delivery of macromolecular compounds such as proteins, DNA, siRNAs, and drug carriers. It is characterized by the unique cell- (or receptor-), temperature-, and payload-independent mechanisms, therefore offering potent means to improve poor cellular uptake of a variety of macromolecular drugs. Nevertheless, thi...

  12. Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells.

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    Alberto Muñoz

    Full Text Available The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW and PAF96 (RKKAAA, were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i its interaction with the cell envelope; (ii its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the

  13. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

  14. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

    Science.gov (United States)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

    2016-11-01

    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  15. Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy.

    Science.gov (United States)

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A; Williams, Donna L; Deuss, Peter; Gait, Michael J

    2015-02-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening. PMID:25412073

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

    Science.gov (United States)

    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.

  17. Thermodynamics of cell-penetrating HIV1 TAT peptide insertion into PC/PS/CHOL model bilayers through transmembrane pores: the roles of cholesterol and anionic lipids.

    Science.gov (United States)

    Hu, Yuan; Patel, Sandeep

    2016-08-10

    Efficient delivery of pharmaceutically active molecules across cellular membranes using cell penetrating peptides (CPPs), such as the cationic human immunodeficiency virus-1 trans-acting activator of transcription peptide (HIV-1 TAT), continues to attract scientific attention in drug design and disease treatment. Experimental results show that the TAT peptide is not only capable of directly penetrating the biological membrane in a passive manner, but also forming physical, membrane-spanning pores that may facilitate transport. Experiments further show that anionic lipids accelerate peptide permeation within a range of mole percentage composition. In this work, we explored the structures and translocation thermodynamics of the cationic TAT peptide across a series of DPPC/DPPS model membranes with the presence of 0-30 mol% cholesterol. We computed the potentials of the mean force by using umbrella sampling molecular dynamics simulations coupled to the Martini coarse-grained force field. We systematically investigated the roles of cholesterol and anionic lipids (membrane surface charge) in TAT peptide translocation. In qualitative agreement with experimental findings, the barrier heights were significantly reduced in the presence of anionic lipids. A toroidal hydrophilic pore was strongly suggested by membrane structure analysis. Cholesterol stabilizes the liquid-ordered (Lo) phase of membranes and increases the elastic stiffness of bilayers. Consequently, it hinders transmembrane pore formation and thus modulates solute permeability, since the liquid-ordered phase suppresses reorientation of the lipid molecules on simulation time scales. Though cholesterol contributes marginally to the total free energy associated with peptide permeation, the coordination of cholesterol to the peptide weakens more favorable peptide-lipid interactions. The addition of the anionic lipid DPPS to the neutral DPPC bilayer leads to the emergence and further enhancement of an interfacially

  18. The feasibility of a targeted ultrasound contrast agent carrying genes and cell-penetrating peptides to hypoxic HUVEC

    International Nuclear Information System (INIS)

    Objective: To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides (CPP) and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells (HUVEC). Methods: Anti-P-selectin targeted ultrasound contrast agent carrying a green fluorescent protein gene (pEGFP-N1) and CPP was prepared by mechanical vibration and carbodiimide techniques. The appearance, distribution, concentration and diameter of the ultrasound contrast agent were measured. The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy (CLSM). The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry. HUVEC were cultured in vitro and hypoxic HUVEC were prepared using hydrogen peroxide (H2O2). Hypoxic HUVEC were randomly assigned targeted ultrasound contrast agents and non-targeted ultrasound contrast agents for transfection. The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry. T-test and linear correlation analysis were used for statistical analysis. Results: The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was (2.15 ±0.36) μm and the concentration was (1.58 ± 0.23) × 107/ml.The results of CLSM showed that gene and CPP were distributed on the shell of the agent. The gene encapsulation efficiency was 28% (y=0.932x-0.09, r=0.993, P<0.05), and the CPP encapsulation efficiency was 25% (y=5.875x-0.81, r=0.987, P<0.05). EGFP expression was observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted ultrasound contrast agents. The average transfection efficiencies of targeted ultrasound contrast agents and non-targeted ultrasound contrast agents were (18.74 ± 0.47) % and (15.34 ± 0.22) % after 24 h (t=10.923, P<0.001). Conclusions: The in vitro studies showed

  19. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    Science.gov (United States)

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  20. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    NARCIS (Netherlands)

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A.; Williams, Donna L.; Deuss, Peter; Gait, Michael J.

    2015-01-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELecti

  1. Targeting the EGFR/PCNA signaling suppresses tumor growth of triple-negative breast cancer cells with cell-penetrating PCNA peptides.

    Directory of Open Access Journals (Sweden)

    Yung-Luen Yu

    Full Text Available Tyrosine 211 (Y211 phosphorylation of proliferation cell nuclear antigen (PCNA coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR tyrosine kinase inhibitor (TKI-resistant cells, both nuclear EGFR (nEGFR expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC. Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP, which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC.

  2. Folic Acid-Targeted and Cell Penetrating Peptide-Mediated Theranostic Nanoplatform for High-Efficiency Tri-Modal Imaging-Guided Synergistic Anticancer Phototherapy.

    Science.gov (United States)

    Li, Na; Li, Tingting; Liu, Chen; Ye, Shiyi; Liang, Jiangong; Han, Heyou

    2016-05-01

    A novel nanomaterial with precisely-defined size and shape, biocompatible composition, and excellent stability, which can integrate multi modal targeted imaging and therapy into a single system for visualized therapeutics, has recently attracted significant research interest. Here, we developed a multifunctional nanoplatform based on silica-coated 4-mercaptobenzoic acid-modified gold nanorods (Au NRs) decorated with gold nanoclusters rich in the photosensitizer Ce6 (Au-Ce6 NCs). The nanoparticles also comprised folic acid and cell penetrating peptide molecules anchored on the surface, obtaining the Au@SiO2@Au-cell penetrating peptide nanocomposite. The Au-Ce6 NCs enhanced the photophysical stability, provided numerous bonding sites and offered a large surface-area and interior space to achieve a high drug loading efficiency (up to 55%). The anchored folic acid and cell penetrating peptide synergistically enhanced the efficiency of uptake of nanocomposites by HeLa cells (up to 70.7%) and improved therapeutic efficacy. The nanocomposite also has good water-solubility, excellent biocompatibility, and long-term stability against illumination and exposure to pH 3-12, thus facilitating their bioapplications in cancer theranostics. Here, the nanocomposite was established for high-resolution and noninvasive tri-modal surface-enhanced Raman spectrum/dark-field/fluorescence imaging-guided high-efficiency synergistic photodynamic/photothermal therapy of cancer. Our studies demonstrate that the multifunctional nanocomposite has the potential as a novel and sensitive contrast agent for complementary and synergistic theranostics in the clinic. PMID:27305812

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

    Science.gov (United States)

    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. PMID:27532224

  4. Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity

    Directory of Open Access Journals (Sweden)

    Lee JY

    2015-08-01

    Full Text Available Jue Yeon Lee,1,* Jin Sook Suh,2,* Jung Min Kim,1 Jeong Hwa Kim,1 Hyun Jung Park,1 Yoon Jeong Park,1,2 Chong Pyoung Chung1 1Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Chungcheongbuk-do, Republic of Korea; 2Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Human beta-defensins (hBDs are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to

  5. Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy.

    Science.gov (United States)

    Jing, Hui; Cheng, Wen; Li, Shouqiang; Wu, Bolin; Leng, Xiaoping; Xu, Shouping; Tian, Jiawei

    2016-10-01

    The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy. PMID:27388967

  6. Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid-polyethylene glycol nanoparticles improves ocular drug delivery

    Directory of Open Access Journals (Sweden)

    Vasconcelos A

    2015-01-01

    Full Text Available Aimee Vasconcelos,1 Estefania Vega,2 Yolanda Pérez,3 María J Gómara,1 María Luisa García,2 Isabel Haro1 1Unit of Synthesis and Biomedical Applications of Peptides, Department of Biomedical Chemistry, Institute for Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC-CSIC, 2Department of Physical Chemistry, Institute of Nanoscience and Nanotechnology, Faculty of Pharmacy, University of Barcelona, 3Nuclear Magnetic Resonance Unit, IQAC-CSIC, Barcelona, Spain Abstract: In this work, a peptide for ocular delivery (POD and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid (PGLA–polyethylene glycol (PEG-nanoparticles (NPs in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide; the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation

  7. Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

    Institute of Scientific and Technical Information of China (English)

    Liping Yang; Yongming Yao; Zhiyong Sheng; Xiaomei Zhu; Yong Jiang

    2009-01-01

    Objective p38 Mitogen-activated protein kinase (MAPK) is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus (HIV)-1 transactivator of transcription (TAT), which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor (ATF) 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

  8. Membrane damage as first and DNA as the secondary target for anti-candidal activity of antimicrobial peptide P7 derived from cell-penetrating peptide ppTG20 against Candida albicans.

    Science.gov (United States)

    Li, Lirong; Song, Fengxia; Sun, Jin; Tian, Xu; Xia, Shufang; Le, Guowei

    2016-06-01

    P7, a peptide analogue derived from cell-penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti-Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l-phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin-treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC-P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27197902

  9. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

    Directory of Open Access Journals (Sweden)

    Ding Y

    2015-10-01

    Full Text Available Yuan Ding,1,* Dan Sun,1,* Gui-Ling Wang,1 Hong-Ge Yang,1 Hai-Feng Xu,1 Jian-Hua Chen,2 Ying Xie,1,3 Zhi-Qiang Wang4 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing, 2School of Medicine, Jianghan University, Wuhan, 3State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, People’s Republic of China; 4Department of Chemistry and Biochemistry, Kent State University Geauga, Burton, OH, USA *These authors contributed equally to this work Abstract: Cell-penetrating peptides (CPPs as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into

  10. Antimicrobial and cell-penetrating properties of penetratin analogs

    DEFF Research Database (Denmark)

    Bahnsen, Jesper Søborg; Franzyk, Henrik; Sandberg-Schaal, Anne;

    2013-01-01

    Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as...

  11. Prolongation of life in rats with malignant glioma by intranasal siRNA/drug codelivery to the brain with cell-penetrating peptide-modified micelles.

    Science.gov (United States)

    Kanazawa, Takanori; Morisaki, Kazuki; Suzuki, Shohei; Takashima, Yuuki

    2014-05-01

    New therapeutic strategies are required to develop candidate drugs and ensure efficient delivery of these drugs to the brain and the central nervous system (CNS). Small interfering RNA (siRNA)-based therapies have been investigated as potential novel approaches for the treatment of brain disorders. Previously, we showed that Tat, a cell-penetrating peptide derived from HIV-Tat, and the modified block copolymers (MPEG-PCL-Tat) can form stable complexes with siRNA or can be loaded with an anticancer drug and efficiently deliver the drugs to the brain tissue via intranasal delivery. In this study, to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, we used MPEG-PCL-Tat micelles with a nose-to-brain delivery system to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) codelivery system. MPEG-PCL-Tat and CPT-loaded MPEG-PCL-Tat can form a stable complex with siRNA with a particle size from 60 to 200 nm and a positive charge at N/P ratios up to 5. Additionally, MPEG-PCL-Tat/siRaf-1 and CPT-loaded MPEG-PCL-Tat/siRaf-1 have fostered cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG-PCL-Tat carrier. Furthermore, compared to the unloaded MPEG-PCL-Tat/siRaf-1 complex, a CPT-loaded MPEG-PCL-Tat/siRaf-1 complex achieved the high therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA codelivery using MPEG-PCL-Tat nanomicelles with nose-to-brain delivery is an excellent therapeutic approach for brain and CNS diseases.

  12. Cellular penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, Bart [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Hu, Meiduo [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); McLarty, Kristin [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Costantini, Dan [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Toronto General Research Institute, University Health Network, Toronto, ON, M5S 3M2 (Canada)]. E-mail: raymond.reilly@utoronto.ca

    2007-01-15

    Introduction: Our objective was to compare the cell penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled immunoconjugates (ICs) composed of 16-mer peptides (GRKKRRQRRRPPQGYG) derived from HIV-1 transactivator of transcription (tat) protein and anti-mouse IgG (mIgG) in BT-474 breast cancer (BC) cells. Methods: [{sup 111}In]tat ICs were constructed by site-specific conjugation of tat peptides to NaIO{sub 4} {sup -}-oxidized carbohydrates in the Fc domain of diethylenetriaminepentaacetic-acid-modified anti-mIgG antibodies. Immunoreactivity against mIgG was assessed in a competition assay. The kinetics of the accumulation of [{sup 111}In]anti-mIgG-tat IC and [{sup 123}I]anti-mIgG-tat ICs in BT-474 cells and the elimination of radioactivity from cells, cytoplasm or nuclei were determined. The effects of excess tat peptides or NH{sub 4}Cl (an inhibitor of endosomal acidification) on cellular uptake and nuclear importation of [{sup 111}In]anti-mIgG-tat were measured. Results: [{sup 111}In]anti-mIgG-tat was >97% radiochemically pure and exhibited preserved immunoreactivity with mIgG epitopes. [{sup 123}I]Anti-mIgG-tat penetrated BT-474 cells more rapidly than [{sup 111}In]anti-mIgG-tat ICs and achieved a 1.5-fold to a 2-fold higher uptake in cells and nuclei. Cell penetration and nuclear uptake of [{sup 111}In]anti-mIgG-tat were inhibited by excess tat peptides and NH{sub 4}Cl. Elimination of radioactivity from BT-474 cells and nuclei was more rapid and complete for {sup 123}I-labeled than for {sup 111}In-labeled anti-mIgG-tat ICs. Conclusion: Tat peptides derived from HIV-1 tat protein promoted the penetration and nuclear uptake of radioactivity following the incubation of {sup 111}In-labeled and {sup 123}I-labeled anti-mIgG antibodies with BT-474 human BC cells. {sup 111}In-labeled tat ICs are feasible for inserting radionuclides into cancer cells with potential for targeting intracellular and, particularly, nuclear epitopes for

  13. Biological activities of selected peptides: skin penetration ability of copper complexes with peptides.

    Science.gov (United States)

    Mazurowska, Lena; Mojski, Miroslaw

    2008-01-01

    This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or gamma-glutamyl-cysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion. PMID:18350235

  14. Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency

    Directory of Open Access Journals (Sweden)

    Howell Stephen B

    2011-02-01

    Full Text Available Abstract Background Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE, which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel to the cytoplasm of the human ovarian carcinoma cell line 2008. Results CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9 was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9 through a G4S linker to R9-rGel (E9-G4S-R9-rGel largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel. Conclusion We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative

  15. 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.

    Science.gov (United States)

    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

  16. Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide.

    Science.gov (United States)

    Wei, Baojun; Wei, Yuxiang; Zhang, Kuo; Wang, Jing; Xu, Ruihuan; Zhan, Sien; Lin, Guigao; Wang, Wei; Liu, Min; Wang, Lunan; Zhang, Rui; Li, Jinming

    2009-05-01

    RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5'-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide. PMID:18823738

  17. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon-Jeong, E-mail: parkyj@snu.ac.kr [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Dental Regenerative Biotechnology, Dental Research Institute and School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: ccpperio@snu.ac.kr [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, College of Dentistry, Seoul National University, Seoul (Korea, Republic of)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  18. Tumor-Penetrating Peptide-Modified DNA Tetrahedron for Targeting Drug Delivery.

    Science.gov (United States)

    Xia, Zhiwei; Wang, Ping; Liu, Xunwei; Liu, Ting; Yan, Yinan; Yan, Juan; Zhong, Jian; Sun, Gang; He, Dannong

    2016-03-01

    DNA self-assembling nanostructure has been considered as a promising candidate as a drug delivery vehicle because of its compactness, mechanical stability, and noncytotoxicity. In this work, we developed functional, multiform DNA nanostructures by appending a tumor-penetrating peptide to tetrahedral DNA nanostructure (p-TDN). This functional structure is able to efficiently increase the rate of uptake of glioblastoma cell U87MG compared with the DNA tetrahedron and the double-stranded DNA structures. We found that the DNA tetrahedron plays the main role in the endocytosis of U87MG cells, whereas the tumor-penetrating peptide could also bind to transmembrane glycoprotein neuropilin-1 and mediate the endocytosis of the p-TDN nanostructure. Moreover, given the high efficiency of the growth inhibitory effect of the p-TDN loading doxorubicin hydrochloride, the p-TDN distinguishes itself as a promising candidate as an effective delivery carrier.

  19. Effects of Sizes and Conformations of Fish-Scale Collagen Peptides on Facial Skin Qualities and Transdermal Penetration Efficiency

    OpenAIRE

    Huey-Jine Chai; Jing-Hua Li; Han-Ning Huang; Tsung-Lin Li; Yi-Lin Chan; Chyuan-Yuan Shiau; Chang-Jer Wu

    2010-01-01

    Fish-scale collagen peptides (FSCPs) were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromis sp.) scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the...

  20. Comparison of mechanisms and cellular uptake of cell-penetrating peptide on different cell lines%不同细胞系对细胞穿透肽的摄取和机制比较

    Institute of Scientific and Technical Information of China (English)

    马冬旭; 齐宪荣

    2010-01-01

    细胞穿透肽(cell-penetrating peptide,CPP)作为一种潜在的药物输送高效转运载体一直得到研究者的广泛关注.本文中采用4种肿瘤细胞系(MCF-7、MDA-MB-231、C6和B16F10)分别摄取异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的CPP,观察到CPP入胞,并具有时间和浓度的依赖性,同时发现了C6细胞对CPP的胞吐作用,其胞吐动力学符合零级方程;在低温(4℃)和内吞抑制剂存在条件下探讨了CPP入胞的机制.低温条件对CPP的入胞未产生抑制作用;肝素钠作为细胞表面硫酸糖蛋白受体抑制剂对CPP的入胞有较强抑制作用,肝素组对CPP的摄取只达到对照组的3%~10%;而氯丙嗪、氯喹和N-乙酰基-N-异丙基阿米洛利[5-(N-ethyL-N-isopropyl)-amiloride,EIPA]对CPP的入胞影响不大.本研究表明,CPP穿透细胞没有选择性,即缺乏细胞特异性,但CPP的摄取量与细胞种类有关.硫酸蛋白聚糖的吸附介导在CPP穿透细胞中发挥了重要作用.

  1. Destruction of amyloid fibrils by graphene through penetration and extraction of peptides.

    Science.gov (United States)

    Yang, Zaixing; Ge, Cuicui; Liu, Jiajia; Chong, Yu; Gu, Zonglin; Jimenez-Cruz, Camilo A; Chai, Zhifang; Zhou, Ruhong

    2015-11-28

    Current therapies for Alzheimer's disease (AD) can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. As such, novel approaches for AD treatment and prevention are urgently needed. We here provide both experimental and computational evidence that pristine graphene and graphene-oxide nanosheets can inhibit Aβ peptide monomer fibrillation and clear mature amyloid fibrils, thus impacting the central molecular superstructures correlated with AD pathogenesis. Our molecular dynamics simulations for the first time reveal that graphene nanosheets can penetrate and extract a large number of peptides from pre-formed amyloid fibrils; these effects seem to be related to exceptionally strong dispersion interactions between peptides and graphene that are further enhanced by strong π-π stacking between the aromatic residues of extracted Aβ peptides and the graphene surface. Atomic force microscopy images confirm these predictions by demonstrating that mature amyloid fibrils can be cut into pieces and cleared by graphene oxides. Thioflavin fluorescence assays further illustrate the detailed dynamic processes by which graphene induces inhibition of monomer aggregation and clearance of mature amyloid fibrils, respectively. Cell viability and ROS assays indicate that graphene oxide can indeed mitigate cytotoxicity of Aβ peptide amyloids. Our findings provide new insights into the underlying molecular mechanisms that define graphene-amyloid interaction and suggest that further research on nanotherapies for Alzheimer's and other protein aggregation-related diseases is warranted.

  2. Intranasal Delivery of Cell-Penetrating anti-NF-κB Peptides (Tat-NBD) Alleviates Infection-Sensitized Hypoxic-Ischemic Brain Injury

    OpenAIRE

    Yang, Dianer; Sun, Yu-Yo; Lin, Xiaoyi; Baumann, Jessica M.; Dunn, R. Scott; Lindquist, Diana M.; Kuan, Chia-Yi

    2013-01-01

    Perinatal infection aggravates neonatal hypoxic-ischemic (HI) brain injury and may interfere with therapeutic hypothermia. While the NF-κB signaling pathway has been implicated in microglia activation in infection-sensitized HI, the current therapeutic strategies rely on systemic intervention, which could impair neonatal immunity and increase the risk of severe infection. To devise a brain-targeted anti-NF-κB strategy, we examined the effects of intranasal delivery of tat-NBD peptides in two ...

  3. Destruction of amyloid fibrils by graphene through penetration and extraction of peptides

    Science.gov (United States)

    Yang, Zaixing; Ge, Cuicui; Liu, Jiajia; Chong, Yu; Gu, Zonglin; Jimenez-Cruz, Camilo A.; Chai, Zhifang; Zhou, Ruhong

    2015-11-01

    Current therapies for Alzheimer's disease (AD) can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. As such, novel approaches for AD treatment and prevention are urgently needed. We here provide both experimental and computational evidence that pristine graphene and graphene-oxide nanosheets can inhibit Aβ peptide monomer fibrillation and clear mature amyloid fibrils, thus impacting the central molecular superstructures correlated with AD pathogenesis. Our molecular dynamics simulations for the first time reveal that graphene nanosheets can penetrate and extract a large number of peptides from pre-formed amyloid fibrils; these effects seem to be related to exceptionally strong dispersion interactions between peptides and graphene that are further enhanced by strong π-π stacking between the aromatic residues of extracted Aβ peptides and the graphene surface. Atomic force microscopy images confirm these predictions by demonstrating that mature amyloid fibrils can be cut into pieces and cleared by graphene oxides. Thioflavin fluorescence assays further illustrate the detailed dynamic processes by which graphene induces inhibition of monomer aggregation and clearance of mature amyloid fibrils, respectively. Cell viability and ROS assays indicate that graphene oxide can indeed mitigate cytotoxicity of Aβ peptide amyloids. Our findings provide new insights into the underlying molecular mechanisms that define graphene-amyloid interaction and suggest that further research on nanotherapies for Alzheimer's and other protein aggregation-related diseases is warranted.Current therapies for Alzheimer's disease (AD) can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. As such, novel approaches for AD treatment and prevention are urgently needed. We

  4. Effects of Sizes and Conformations of Fish-Scale Collagen Peptides on Facial Skin Qualities and Transdermal Penetration Efficiency

    Directory of Open Access Journals (Sweden)

    Huey-Jine Chai

    2010-01-01

    Full Text Available Fish-scale collagen peptides (FSCPs were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromis sp. scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the lighter FSCPs, 2000 and 1300 Da. In addition, the heavier seemed to preserve favorable coiled structures comparing to the lighter that presents mainly as linear under confocal scanning laser microscopy. FSCPs, particularly the heavier, were concluded to efficiently penetrate stratum corneum to epidermis and dermis, activate fibroblasts, and accelerate collagen synthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preserving the given desired structure feature.

  5. Effects of sizes and conformations of fish-scale collagen peptides on facial skin qualities and transdermal penetration efficiency.

    Science.gov (United States)

    Chai, Huey-Jine; Li, Jing-Hua; Huang, Han-Ning; Li, Tsung-Lin; Chan, Yi-Lin; Shiau, Chyuan-Yuan; Wu, Chang-Jer

    2010-01-01

    Fish-scale collagen peptides (FSCPs) were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromis sp.) scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the lighter FSCPs, 2000 and 1300 Da. In addition, the heavier seemed to preserve favorable coiled structures comparing to the lighter that presents mainly as linear under confocal scanning laser microscopy. FSCPs, particularly the heavier, were concluded to efficiently penetrate stratum corneum to epidermis and dermis, activate fibroblasts, and accelerate collagen synthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preserving the given desired structure feature. PMID:20625414

  6. Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems

    Directory of Open Access Journals (Sweden)

    Sara Trabulo

    2010-03-01

    Full Text Available The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides. In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.

  7. Penetration of Milk-Derived Antimicrobial Peptides into Phospholipid Monolayers as Model Biomembranes

    Directory of Open Access Journals (Sweden)

    Wanda Barzyk

    2013-01-01

    Full Text Available Three antimicrobial peptides derived from bovine milk proteins were examined with regard to penetration into insoluble monolayers formed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol sodium salt (DPPG. Effects on surface pressure (Π and electric surface potential (ΔV were measured, Π with a platinum Wilhelmy plate and ΔV with a vibrating plate. The penetration measurements were performed under stationary diffusion conditions and upon the compression of the monolayers. The two type measurements showed greatly different effects of the peptide-lipid interactions. Results of the stationary penetration show that the peptide interactions with DPPC monolayer are weak, repulsive, and nonspecific while the interactions with DPPG monolayer are significant, attractive, and specific. These results are in accord with the fact that antimicrobial peptides disrupt bacteria membranes (negative while no significant effect on the host membranes (neutral is observed. No such discrimination was revealed from the compression isotherms. The latter indicate that squeezing the penetrant out of the monolayer upon compression does not allow for establishing the penetration equilibrium, so the monolayer remains supersaturated with the penetrant and shows an under-equilibrium orientation within the entire compression range, practically.

  8. 经穿膜肽与PEG修饰的核糖体失活蛋白Gelonin抗肿瘤作用的研究%Study on cell-penetrating peptide modified and PEGylated ribosome inactive protein Gelonin

    Institute of Scientific and Technical Information of China (English)

    张娅洁; 王慧媛; 陈应之; 汤懿斯; 杨志民; 黄永焯

    2015-01-01

    Objective:To improve anti-tumor effect of Gelonin, the plant-sourced RIP is modified by chemically conjugating a cell-penetrating peptide and polyethylene glycol (PEG). Methods:Purified protein was obtained after being performed on FPLC (fast protein liquid chromatography) Superdex75 column. Cytotoxicity was detected by MTT assay. The cellular uptake by HT1080 cells was studied by using inverted fluorescence microscopy and flow cytometry. In-vivo imaging technology was utilized for investigation of the in-vivo drug distribution in the HT1080 tumor-bearing mice. Results:The modified product was purified by using gel filtration chromatergraphy. Moreover, compared with native Gelonin, the cytotoxicity of modified protein was increased, especially in HT1080, presumably due to the enhanced cellular uptake. The in-vivo imaging results suggested that drug accumulation in tumor was improved by PEGylation. Conclusion:Modified Gelonin can improve cell penetration and cytotoxicity in tumor cells. PEGylation can increase tumor accumulation of the protein drug, and thereby enhance its anti-tumor effect.%目的:通过对核糖体失活蛋白Gelonin进行化学修饰,利用穿膜肽和聚乙二醇(PEG)偶联来提高其到达肿瘤部位和进入肿瘤细胞的能力,使Gelonin更高效地发挥抑瘤作用. 方法:利用FPLC Superdex75分子筛预装柱纯化系统对所修饰的Gelonin进行纯化后,在不同细胞系测试细胞毒性;通过倒置荧光显微镜、流式细胞分析技术等对药物进入纤维肉瘤细胞HT1080的能力进行评价;采用小动物活体成像技术考察药物体系在荷瘤动物体内的分布情况. 结果:采用分子筛色谱纯化可以得到纯度相对较高的修饰产物,其毒性较无修饰的Gelonin强,且在HT1080细胞系作用最明显;细胞摄取结果显示,与未修饰的Gelonin相比,该药物体系具有更高的细胞摄取效率;动物成像结果表明,PEG5000修饰可以改变Gelonin在动物体内的分布情况,

  9. Market penetration scenarios for fuel cell vehicles

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, C.E.; James, B.D.; Lomax, F.D. Jr. [Directed Technologies, Inc., Arlington, VA (United States)

    1997-12-31

    Fuel cell vehicles may create the first mass market for hydrogen as an energy carrier. Directed Technologies, Inc., working with the US Department of Energy hydrogen systems analysis team, has developed a time-dependent computer market penetration model. This model estimates the number of fuel cell vehicles that would be purchased over time as a function of their cost and the cost of hydrogen relative to the costs of competing vehicles and fuels. The model then calculates the return on investment for fuel cell vehicle manufacturers and hydrogen fuel suppliers. The model also projects the benefit/cost ratio for government--the ratio of societal benefits such as reduced oil consumption, reduced urban air pollution and reduced greenhouse gas emissions to the government cost for assisting the development of hydrogen energy and fuel cell vehicle technologies. The purpose of this model is to assist industry and government in choosing the best investment strategies to achieve significant return on investment and to maximize benefit/cost ratios. The model can illustrate trends and highlight the sensitivity of market penetration to various parameters such as fuel cell efficiency, cost, weight, and hydrogen cost. It can also illustrate the potential benefits of successful R and D and early demonstration projects. Results will be shown comparing the market penetration and return on investment estimates for direct hydrogen fuel cell vehicles compared to fuel cell vehicles with onboard fuel processors including methanol steam reformers and gasoline partial oxidation systems. Other alternative fueled vehicles including natural gas hybrids, direct injection diesels and hydrogen-powered internal combustion hybrid vehicles will also be analyzed.

  10. Skin Penetrating Peptide as a Tool to Enhance the Permeation of Heparin through Human Epidermis.

    Science.gov (United States)

    Gennari, Chiara G M; Franzè, Silvia; Pellegrino, Sara; Corsini, Emanuela; Vistoli, Giulio; Montanari, Luisa; Minghetti, Paola; Cilurzo, Francesco

    2016-01-11

    This study aimed to identify a new skin penetrating peptide (SPP) able to enhance unfractionated heparin (UFH) permeation through human epidermis by screening a phage display peptide library. The effects of the synthesized heptapeptide (DRTTLTN) on human stratum corneum organization were investigated by ATR-FTIR spectroscopy and molecular dynamics simulation. The DRTTLTN penetration within the human epidermis caused both a fluidization of the stratum corneum lipids and the extension of keratins due to the increase of the contribution of α-helices. The coadministration of DRTTLTN with UFH resulted ineffective in increasing skin penetration due to UFH affinity for keratins. The conjugation of DRTTLTN to UFH by N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride and sodium N-hydroxysulfosuccinimide led to an increase of the flux of 24-36-fold with respect to raw UFH, depending on the adopted synthetic procedure. The new compounds showed a decrease of the antifactor Xa activity of about 4-5 times. DRTTLTN also permitted to increase the fluxes of small model molecules. In conclusion, these data support the use of SPP to enhance the skin penetration of poorly absorbed compounds even in the case of macromolecules as polysaccharides. PMID:26623948

  11. SAP(E) - A cell-penetrating polyproline helix at lipid interfaces.

    Science.gov (United States)

    Franz, Johannes; Lelle, Marco; Peneva, Kalina; Bonn, Mischa; Weidner, Tobias

    2016-09-01

    Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides - SAP(E) - which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly - since peptide-membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur.

  12. SAP(E) - A cell-penetrating polyproline helix at lipid interfaces.

    Science.gov (United States)

    Franz, Johannes; Lelle, Marco; Peneva, Kalina; Bonn, Mischa; Weidner, Tobias

    2016-09-01

    Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides - SAP(E) - which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly - since peptide-membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur. PMID:27237727

  13. 穿膜肽引导的体外表达转录因子蛋白Sox2进入红鳍东方鲀精巢细胞系%Intracellular delivery of the expressed fusion protein Sox2 with cell penetrating peptides to cultured Takifugu rupies spermary cells

    Institute of Scientific and Technical Information of China (English)

    杨秀霞; 侯雪宁; 徐彬; 郝萧; 姜国建; 樊廷俊

    2014-01-01

    为了探索适用于体外培养的鱼细胞外源基因转入方法,本研究通过构建红鳍东方鲀(Takifugu rubripes)转录因子Sox2的重组表达载体 pET32a(+)-Sox2-11R-6His,诱导表达并纯化得到了 C末端连接多聚精氨酸(11R)的重组蛋白Sox2-11R-6His,以其与红鳍东方鲀精巢细胞系细胞共孵育12 h后,光镜观察结合Western Blot检测发现重组蛋白进入细胞的效率与浓度呈剂量依赖关系且最佳孵育浓度为8μg/mL,当重组蛋白质量浓度达到10μg/mL 时,表现出明显的细胞毒性。对外源蛋白进行免疫荧光标记定位,发现重组蛋白分布于细胞质,部分进入到细胞核中。证明了穿膜肽11R可以有效运载转录因子重组蛋白至红鳍东方鲀的细胞系细胞中。本研究旨在将广泛应用于哺乳动物的细胞基因递送载体穿膜肽应用于鱼类细胞系细胞。%Research on exogenous gene function in cultured cell line cells is guaranteed by two biological processes, efficient gene transduction and errorless gene expression. Fish cells are not easy for exogenous gene transduction and expression depending on the gene delivery method used and the transgene promoter. Cell penetrating peptides (CPPs), such as the CPP of the human immunodeficiency virus type 1 TAT protein, poly-arginine (6–12 residues), and the CPP derived from flock house virus (FHV), are short cationic and/or amphipathic peptides that are able to transport various biological compounds such as peptides, proteins, and oligonucleotides into mammalian cells to modulate biological activities inside cells. Although it has been shown that CPPs can mediate efficient delivery into a wide variety of mam-malian cell types, the transduction of proteins to cultured fish cells is considered more challenging. In this work, the Sox2 gene from Takifugu rupies genomic DNA was cloned and subsequent expression was carried out in Escherichia coli in the form of recombinant proteins by introducing cell

  14. Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

    Science.gov (United States)

    Sha, Huizi; Zou, Zhengyun; Xin, Kai; Bian, Xinyu; Cai, Xueting; Lu, Wuguang; Chen, Jiao; Chen, Gang; Huang, Leaf; Blair, Andrew M.; Cao, Peng; Liu, Baorui

    2016-01-01

    Human tumors, including gastric cancer, frequently express high levels of epidermal growth factor receptors (EGFRs), which are associated with a poor prognosis. Targeted delivery of anticancer drugs to cancerous tissues shows potential in sparing unaffected tissues. However, it has been a major challenge for drug penetration in solid tumor tissues due to the complicated tumor microenvironment. We have constructed a recombinant protein named anti-EGFR-iRGD consisting of an anti-EGFR VHH (the variable domain from the heavy chain of the antibody) fused to iRGD, a tumor-specific binding peptide with high permeability. Anti-EGFR-iRGD, which targets EGFR and αvβ3, spreads extensively throughout both the multicellular spheroids and the tumor mass. The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines, multicellular spheroids, and mice. Moreover, anti-EGFR-iRGD could improve anticancer drugs, such as doxorubicin (DOX), bevacizumab, nanoparticle permeability and efficacy in multicellular spheroids. This study draws attention to the importance of iRGD peptide in the therapeutic approach of anti-EGFR-iRGD. As a consequence, anti-EGFR-iRGD could be a drug candidate for cancer treatment and a useful adjunct of other anticancer drugs. PMID:25553823

  15. Discovery and characterization of a new cell-penetrating protein.

    Science.gov (United States)

    Simeon, Rudo L; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei

    2013-12-20

    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.

  16. Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

    Science.gov (United States)

    Wang, Ting-Yi; Pellois, Jean-Philippe

    2016-01-01

    Cell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined. This in turn has hampered their usage in biotechnological or therapeutic applications. We have recently reported that the cell penetration activity of poly-arginine CPPs (PACPPs) is dependent on the oxidation status of the plasma membrane of cells. Our data support a model where the positively-charged PACPP binds negatively-charged lipids exposed on the cell surface as a result of oxidative damage. The PACPP then crosses the membrane via formation of inverted micelles with these anionic lipids. This model provides a plausible explanation for the high variability in the cell delivery efficiency of a PACPP often observed in different settings. Notably, taking into account the current literature describing the effects of lipid oxidation, our data point to a highly complex and underappreciated interplay between PACPPs and oxidized membrane species. Overall, a better understanding of oxidation-dependent cell penetration might provide a fundamental basis for development of optimal cell permeable peptides (including cyclic peptides, stapled peptides, peptoids, etc…) and of robust delivery protocols.

  17. Investigation of penetration force of living cell using an atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Eun Young; Kim, Young Tae; Kim, Dae Eun [Yonsei University, Seoul (Korea, Republic of)

    2009-07-15

    Recently, the manipulation of a single cell has been receiving much attention in transgenesis, in-vitro fertilization, individual cell based diagnosis, and pharmaceutical applications. As these techniques require precise injection and manipulation of cells, issues related to penetration force arise. In this work the penetration force of living cell was studied using an atomic force microscope (AFM). L929, HeLa, 4T1, and TA3 HA II cells were used for the experiments. The results showed that the penetration force was in the range of 2{approx}22 nN. It was also found that location of cell penetration and stiffness of the AFM cantilever affected the penetration force significantly. Furthermore, double penetration events could be detected, due to the multi-membrane layers of the cell. The findings of this work are expected to aid in the development of precision micro-medical instruments for cell manipulation and treatment

  18. A peptide antagonist disrupts NK cell inhibitory synapse formation.

    Science.gov (United States)

    Borhis, Gwenoline; Ahmed, Parvin S; Mbiribindi, Bérénice; Naiyer, Mohammed M; Davis, Daniel M; Purbhoo, Marco A; Khakoo, Salim I

    2013-03-15

    Productive engagement of MHC class I by inhibitory NK cell receptors depends on the peptide bound by the MHC class I molecule. Peptide:MHC complexes that bind weakly to killer cell Ig-like receptors (KIRs) can antagonize the inhibition mediated by high-affinity peptide:MHC complexes and cause NK cell activation. We show that low-affinity peptide:MHC complexes stall inhibitory signaling at the step of Src homology protein tyrosine phosphatase 1 recruitment and do not go on to form the KIR microclusters induced by high-affinity peptide:MHC, which are associated with Vav dephosphorylation and downstream signaling. Furthermore, the low-affinity peptide:MHC complexes prevented the formation of KIR microclusters by high-affinity peptide:MHC. Thus, peptide antagonism of NK cells is an active phenomenon of inhibitory synapse disruption.

  19. Cell-penetrating compounds preferentially bind glycosaminoglycans over plasma membrane lipids in a charge density- and stereochemistry-dependent manner.

    Science.gov (United States)

    Prevette, Lisa E; Benish, Nicolas C; Schoenecker, Amber R; Braden, Kristin J

    2015-12-01

    Cell-penetrating compounds (CPCs) are often conjugated to drugs and genes to facilitate cellular uptake. We hypothesize that the electrostatic interaction between the positively charged amines of the cell-penetrating compounds and the negatively charged glycosaminoglycans (GAGs) extending from cell surfaces is the initiating step in the internalization process. The interactions of generation 5 PAMAM dendrimer, Tat peptide and 25 kDa linear PEI with four different GAGs have been studied using isothermal titration calorimetry to elucidate structure-function relationships that could lead to improved drug and gene delivery methods to a wide variety of cell types. Detailed thermodynamic analysis has determined that CPC-GAG binding constants range from 8.7×10(3) to 2.4×10(6)M(-1) and that affinity is dependent upon GAG charge density and stereochemistry and CPC molecular weight. The effect of GAG composition on affinity is likely due to hydrogen bonding between CPC amines and amides and GAG hydroxyl and amine groups. These results were compared to the association of CPCs with lipid vesicles of varying composition as model plasma membranes to finally clarify the relative importance of each cell surface component in initial cell recognition. CPC-lipid affinity increases with anionic lipid content, but GAG affinity is higher for all cell-penetrating compounds, confirming the role these heterogeneous polysaccharides play in cellular association and clustering.

  20. Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

    OpenAIRE

    Antharam, Vijay C.; Elliott, Douglas W.; Mills, Frank D.; Farver, R. Suzanne; Sternin, Edward; Long, Joanna R.

    2009-01-01

    KL4 is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL4 binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL4 is dep...

  1. Penetration depth of surfactant peptide KL4 into membranes is determined by fatty acid saturation.

    Science.gov (United States)

    Antharam, Vijay C; Elliott, Douglas W; Mills, Frank D; Farver, R Suzanne; Sternin, Edward; Long, Joanna R

    2009-05-20

    KL(4) is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL(4) binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL(4) is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL(4) is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL(4), and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL(4). Additionally, a clear effect of KL(4) on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL(4) causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL(4) to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment. PMID:19450480

  2. Synthetic Peptide libraries for T-cell epitope identification.

    Science.gov (United States)

    Hiemstra, H S; Drijfhout, J W; Koning, F

    2000-01-01

    This chapter describes a methodology for elucidating immunogenic epitopes stimulatory for CD4(+) T-cell clones (Fig. 1). The methodology makes use of synthetic peptide libraries and must be regarded as an alternative to other approaches, such as peptide elution or the application of genetic libraries. The methodology only requires knowledge about the restriction element of the T-cell clone. The restriction element determines which major histocompatibility complex (MHC)-binding anchor motif must be built into the library peptides. A synthetic peptide library is prepared comprising approx 8 million peptides. The synthesis proceeds via a mix-and-split protocol using a solidphase approach on a hybrid resin (1,2). On a hybrid resin, most of the peptide material (84%) is attached via an acid-labile linker whereas the remaining part of the peptide material is acid-stable attached (3). During synthesis, resinbound peptides comprising 14 amino acid residues are produced, with each resin bead containing one unique peptide (4,5). The beads are split into 384 pools, with each pool containing 20,000 beads. From each pool, about 28% of the peptide material is cleaved from every bead. Subsequently, in the first screening round, the 384 pools, each containing 20,000 solubilized peptides, are tested in a proliferation assay with the T-cell clone. Fig. 1. Flow diagram of the complete procedure for the identification of T-cell epitopes using synthetic peptide libraries (1).

  3. RGDC Peptide Modified Quantum Dots Labelling and Imaging of Tumor Cells

    Institute of Scientific and Technical Information of China (English)

    GUO Yi; LI Chun-rong; SHEN Huai-bin; ZHANG Xue-zhong; LI Lin-song; YU Qian; XU Li

    2011-01-01

    The labelling and imaging of tumor cells were investigated via arginine-glycine-aspartic acidcysteine(RGDC) peptide-labelled quantum dots(QDs).The results show that RGDC modified QDs can label SMMC-7721 tumor cells and adhere to cellular membrane.In constrast,the unmodified QDs are mainly dispersed around the cell.We also found that the RGDC-QDs can penetrate into the cell at 2 h of incubation.After 6 h of incubation,RGDC-QDs can accumulate in a unique intracellular region.

  4. Peptides whose uptake by cells is controllable

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Tao; Tsien, Roger Y

    2014-02-04

    A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. Cleavage of X allows separation of A from B, unmasking the normal ability of the basic amino acids in B to drag cargo C into cells near the cleavage event. X is cleaved extracellularly, preferably under physiological conditions. D-amino acids are preferred for the A and B portions, to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases.

  5. Does ligand-receptor mediated competitive effect or penetrating effect of iRGD peptide when co-administration with iRGD-modified SSL?

    Science.gov (United States)

    Zhang, Wei-Qiang; Yu, Ke-Fu; Zhong, Ting; Luo, Li-Min; Du, Ruo; Ren, Wei; Huang, Dan; Song, Ping; Li, Dan; Zhao, Yang; Wang, Chao; Zhang, Xuan

    2015-12-01

    Ligand-mediated targeting of anticancer therapeutic agents is a useful strategy for improving anti-tumor efficacy. It has been reported that co-administration of a tumor-penetrating peptide iRGD (CRGDK/RGPD/EC) enhances the efficacy of anticancer drugs. Here, we designed an experiment involving co-administration of iRGD-SSL-DOX with free iRGD to B16-F10 tumor bearing mice to examine the action of free iRGD. We also designed an experiment to investigate the location of iRGD-modified SSL when co-administered with free iRGD or free RGD to B16-F10 tumor bearing nude mice. Considering the sequence of iRGD, we selected the GPDC, RGD and CRGDK as targeting ligands to investigate the targeting effect of these peptides compared with iRGD on B16-F10 and MCF-7 cells, with or without enzymatic degradation. Finally, we selected free RGD, free CRGDK and free iRGD as ligand to investigate the inhibitory effect on RGD-, CRGDK- or iRGD-modified SSL on B16-F10 or MCF-7 cells. Our results indicated that iRGD targeting to tumor cells was ligand-receptor mediated involving RGD to αv-integrin receptor and CRGDK to NRP-1 receptor. Being competitive effect, the administration of free iRGD would not be able to further enhance the anti-tumor activity of iRGD-modified SSL. There is no need to co-administrate of free iRGD with the iRGD-modified nanoparticles for further therapeutic benefit.

  6. Genetic induction of the gastrin releasing peptide receptor on tumor cells for radiolabeled peptide binding

    International Nuclear Information System (INIS)

    10 and 100 plaque forming units (PFU)/cell and assayed 48 hr later for GRPr expression by 125I-labeled bombesin binding. One-million cells were incubated with 0.1 μCi 125I-labeled bombesin for 1 hr at room temperature and then washed. The percentage binding was determined. Negative controls included untransfected cells or cells transduced with AdCMVLacZ. BNR-11 cells stably transduced to express the mGRPr were used as a positive control. Results: The AdpL results indicated enhanced expression of mGRPr in comparison to background levels measured in uninfected cells or cells transfected with control plasmid encoding the LacZ reporter gene. The binding assay data indicated 26% and 43% binding to HeLa and A427 cells respectively following AdpL transfection. The percentage binding to SKOV3.ip1 and MD-MB-231 cells following AdCMVGRPr transfection with 10 PFU/cell were 14% and 49%, respectively. This increased to 64% and 69% respectively following transfection with 100 PFU/cell which was similar to the 65% binding to BNR-11 stably transfected cells. Thus, we demonstrated the ability to genetically transduce tumor cells to express a novel receptor (mGRPr) targetable by a radiolabeled peptide at a level comparable to a cell line stably expressing the mGRPr. Conclusion: These preliminary in vitro studies demonstrate the ability to transduce tumor cells to express novel receptors targetable by radiolabeled ligands in vitro. This establishes the rationale to further develop this approach for experimental radiotherapy in the context of developing murine animal models to apply this strategy. Our goal is to improve upon conventional RAIT approaches by transducing expression of a new receptor, mGRPr, resulting in high levels of cell surface expression. Furthermore, this strategy will employ radiolabeled low molecular weight peptides rather than antibodies to potentially improve tumor penetration and tumor to normal tissue targeting ratios in vivo

  7. Peptides whose uptake by cells is controllable

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Tao; Olson, Emilia S.; Whitney, Michael; Tsien, Roger

    2015-07-07

    A generic structure for the peptides of the present invention includes A-X-B-C, where C is a cargo moiety, the B portion includes basic amino acids, X is a cleavable linker sequence, and the A portion includes acidic amino acids. The intact structure is not significantly taken up by cells; however, upon extracellular cleavage of X, the B-C portion is taken up, delivering the cargo to targeted cells. Cargo may be, for example, a contrast agent for diagnostic imaging, a chemotherapeutic drug, or a radiation-sensitizer for therapy. X may be cleaved extracellularly or intracellularly. The molecules of the present invention may be linear, cyclic, branched, or have a mixed structure.

  8. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B. (Stanford Univ. School of Medicine, CA (USA) Palo Alto Veterans Administration Medical Center, CA (USA)); Rubin, D.H. (Univ. of Pennsylvania, Philadelphia (USA))

    1988-04-01

    Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

  9. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes

    DEFF Research Database (Denmark)

    Schmidt, Esben G W; Buus, Soren; Thorn, Mette;

    2009-01-01

    in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood...... effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.......Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8(+) T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to...

  10. Interaction of peptides with cell membranes: insights from molecular modeling

    Science.gov (United States)

    Li, Zhen-lu; Ding, Hong-ming; Ma, Yu-qiang

    2016-03-01

    The investigation of the interaction of peptides with cell membranes is the focus of active research. It can enhance the understanding of basic membrane functions such as membrane transport, fusion, and signaling processes, and it may shed light on potential applications of peptides in biomedicine. In this review, we will present current advances in computational studies on the interaction of different types of peptides with the cell membrane. Depending on the properties of the peptide, membrane, and external environment, the peptide-membrane interaction shows a variety of different forms. Here, on the basis of recent computational progress, we will discuss how different peptides could initiate membrane pores, translocate across the membrane, induce membrane endocytosis, produce membrane curvature, form fibrils on the membrane surface, as well as interact with functional membrane proteins. Finally, we will present a conclusion summarizing recent progress and providing some specific insights into future developments in this field.

  11. Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety.

    Science.gov (United States)

    Zhu, Lin; Wang, Tao; Perche, Federico; Taigind, Anton; Torchilin, Vladimir P

    2013-10-15

    In response to the challenges of cancer chemotherapeutics, including poor physicochemical properties, low tumor targeting, insufficient tumor cell internalization/bioavailability, and side effects, we developed a unique tumor-targeted micellar drug-delivery platform. Using paclitaxel as a model therapeutic, a nanopreparation composed of a matrix metalloproteinase 2 (MMP2)-sensitive self-assembly PEG 2000-paclitaxel conjugate (as a prodrug and MMP 2-sensitive moiety), transactivating transcriptional activator peptide-PEG1000-phosphoethanolamine (PE) (a cell-penetrating enhancer), and PEG1000-PE (a nanocarrier building block) was prepared. Several major drug delivery strategies, including self-assembly, PEGylation, the enhanced permeability and retention effect, stimulus sensitivity, a cell-penetrating moiety, and the concept of prodrug, were used in design of this nanoparticle in a collaborative manner. The nanopreparation allowed superior cell internalization, cytotoxicity, tumor targeting, and antitumor efficacy in vitro and in vivo over its nonsensitive counterpart, free paclitaxel and conventional micelles. This uniquely engineered nanoparticle has potential for effective intracellular delivery of drug into cancer cells. PMID:24062440

  12.   Cell Penetrating Peptoids (CPPos: Synthesis of a Small Combinatorial Library by Using IRORI MiniKans

    Directory of Open Access Journals (Sweden)

    Dominik K. Kölmel

    2012-11-01

    Full Text Available Cell penetrating peptoids (CPPos are potent mimics of the corresponding cell penetrating peptides (CPPs. The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.

  13. Peptide pool immunization and CD8+ T cell reactivity

    DEFF Research Database (Denmark)

    Rasmussen, Susanne B; Harndahl, Mikkel N; Buus, Anette Stryhn;

    2013-01-01

    Mice were immunized twice with a pool of five peptides selected among twenty 8-9-mer peptides for their ability to form stable complexes at 37°C with recombinant H-2K(b) (half-lives 10-15h). Vaccine-induced immunity of splenic CD8(+) T cells was studied in a 24h IFNγ Elispot assay. Surprisingly......, IFNγ spot-formation was observed without addition of peptide to the assay culture at 3 weeks and 3 months after immunization. To clarify if IFNγ spot formation in the absence of peptide exposure ex vivo is caused by the peptide-pool per se, mice were immunized with single peptides. Three of the five...

  14. Peptides: A new class of anticancer drugs

    Directory of Open Access Journals (Sweden)

    Ryszard Smolarczyk

    2009-07-01

    Full Text Available Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides, which bind specifically to precise molecular targets located on the surface of tumor cells. Antibacterial peptides bind to both cell and mitochondrial membranes. Some of these peptides attach to the cell membrane, resulting in its disorganization. Other antibacterial peptides penetrate cancer cells without causing cell membrane damage, but they disrupt mitochondrial membranes. Thanks to phage and aptamer libraries, it has become possible to obtain synthetic peptides blocking or activating some target proteins found in cancer cells as well as in cells forming the tumor environment. These synthetic peptides can feature anti-angiogenic properties, block enzymes indispensable for sustained tumor growth, and reduce tumor ability to metastasize. In this review the properties of peptides belonging to both categories are discussed and attempts of their application for therapeutic purposes are outlined.

  15. The Effect of Superparamagnetic Iron Oxide with iRGD Peptide on the Labeling of Pancreatic Cancer Cells In Vitro: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Hou Dong Zuo

    2014-01-01

    Full Text Available The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be transfected into cells using iRGD. Despite the many kinds of cell labeling studies that have been performed with SPIO nanoparticles and RGD peptide or its analogues, only a few have applied SPIO nanoparticles with iRGD peptide in pancreatic cancer cells. This paper reports our preliminary findings regarding the effect of iRGD peptide (CRGDK/RGPD/EC combined with SPIO on the labeling of pancreatic cancer cells. The results suggest that SPIO with iRGD peptide can enhance the positive labeling rate of cells and the uptake of SPIO. Optimal functionalization was achieved with the appropriate concentration or concentration range of SPIO and iRGD peptide. This study describes a simple and economical protocol to label panc-1 cells using SPIO in combination with iRGD peptide and may provide a useful method to improve the sensitivity of pancreatic cancer imaging.

  16. Interaction of peptides with cell membranes: insights from molecular modeling

    International Nuclear Information System (INIS)

    The investigation of the interaction of peptides with cell membranes is the focus of active research. It can enhance the understanding of basic membrane functions such as membrane transport, fusion, and signaling processes, and it may shed light on potential applications of peptides in biomedicine. In this review, we will present current advances in computational studies on the interaction of different types of peptides with the cell membrane. Depending on the properties of the peptide, membrane, and external environment, the peptide–membrane interaction shows a variety of different forms. Here, on the basis of recent computational progress, we will discuss how different peptides could initiate membrane pores, translocate across the membrane, induce membrane endocytosis, produce membrane curvature, form fibrils on the membrane surface, as well as interact with functional membrane proteins. Finally, we will present a conclusion summarizing recent progress and providing some specific insights into future developments in this field. (topical review)

  17. Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV.

    Science.gov (United States)

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Bangphoomi, Kunan; Poungpair, Ornnuthchar; Maneewatch, Santi; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

    2012-01-01

    NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  18. Engineered peptide-based nanobiomaterials for electrochemical cell chip

    Science.gov (United States)

    Kafi, Md. Abdul; Cho, Hyeon-Yeol; Choi, Jeong-Woo

    2016-07-01

    Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly- l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

  19. pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

    Science.gov (United States)

    Kaliaperumal, Jagatheesh; Hari, Natarajan; Pavankumar, Padarthi; Elangovan, Namasivayam

    2016-06-01

    The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H1 NMR. Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

  20. Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery.

    Science.gov (United States)

    McKinlay, Colin J; Waymouth, Robert M; Wender, Paul A

    2016-03-16

    The design, synthesis, and biological evaluation of a new family of highly effective cell-penetrating molecular transporters, guanidinium-rich oligophosphoesters, are described. These unique transporters are synthesized in two steps, irrespective of oligomer length, by the organocatalytic ring-opening polymerization (OROP) of 5-membered cyclic phospholane monomers followed by oligomer deprotection. Varying the initiating alcohol results in a wide variety of cargo attachment strategies for releasable or nonreleasable transporter applications. Initiation of oligomerization with a fluorescent probe produces, upon deprotection, a transporter-probe conjugate that is shown to readily enter multiple cell lines in a dose-dependent manner. These new transporters are superior in cell uptake to previously studied guanidinium-rich oligocarbonates and oligoarginines, showing over 2-fold higher uptake than the former and 6-fold higher uptake than the latter. Initiation with a protected thiol gives, upon deprotection, thiol-terminated transporters which can be thiol-click conjugated to a variety of probes, drugs and other cargos as exemplified by the conjugation and delivery of the model probe fluorescein-maleimide and the medicinal agent paclitaxel (PTX) into cells. Of particular significance given that drug resistance is a major cause of chemotherapy failure, the PTX-transporter conjugate, designed to evade Pgp export and release free PTX after cell entry, shows efficacy against PTX-resistant ovarian cancer cells. Collectively this study introduces a new and highly effective class of guanidinium-rich cell-penetrating transporters and methodology for their single-step conjugation to drugs and probes, and demonstrates that the resulting drug/probe-conjugates readily enter cells, outperforming previously reported guanidinium-rich oligocarbonates and peptide transporters. PMID:26900771

  1. Vaccination against Experimental Allergic Encephalomyelitis with T Cell Receptor Peptides

    Science.gov (United States)

    Howell, Mark D.; Winters, Steven T.; Olee, Tsaiwei; Powell, Henry C.; Carlo, Dennis J.; Brostoff, Steven W.

    1989-11-01

    Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by CD4+ T cells reactive with myelin basic protein (MBP). Rats were rendered resistant to the induction of EAE by vaccination with synthetic peptides corresponding to idiotypic determinants of the β chain VDJ region and Jα regions of the T cell receptor (TCR) that are conserved among encephalitogenic T cells. These findings demonstrate the utility of TCR peptide vaccination for modulating the activity of autoreactive T cells and represent a general therapeutic approach for T cell--mediated pathogenesis.

  2. Trefoil peptides promote restitution of wounded corneal epithelial cells.

    Science.gov (United States)

    Göke, M N; Cook, J R; Kunert, K S; Fini, M E; Gipson, I K; Podolsky, D K

    2001-04-01

    The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

  3. Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity.

    Science.gov (United States)

    Zhang, Shi-Kun; Song, Jin-Wen; Gong, Feng; Li, Su-Bo; Chang, Hong-Yu; Xie, Hui-Min; Gao, Hong-Wei; Tan, Ying-Xia; Ji, Shou-Ping

    2016-01-01

    AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents. PMID:27271216

  4. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    International Nuclear Information System (INIS)

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases

  5. Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin.

    Science.gov (United States)

    Gil-Parrado, Shirley; Assfalg-Machleidt, Irmgard; Fiorino, Ferdinando; Deluca, Dominga; Pfeiler, Dietmar; Schaschke, Norbert; Moroder, Luis; Machleidt, Werner

    2003-03-01

    The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains. PMID:12715890

  6. Mast cells, peptides and cardioprotection - an unlikely marriage?

    LENUS (Irish Health Repository)

    Walsh, S K

    2012-01-31

    1 Mast cells have classically been regarded as the \\'bad guys\\' in the setting of acute myocardial ischaemia, where their released contents are believed to contribute both to tissue injury and electrical disturbances resulting from ischaemia. Recent evidence suggests, however, that if mast cell degranulation occurs in advance of ischaemia onset, this may be cardioprotective by virtue of the depletion of mast cell contents that can no longer act as instruments of injury when the tissue becomes ischaemic. 2 Many peptides, such as ET-1, adrenomedullin, relaxin and atrial natriuretic peptide, have been demonstrated to be cardioprotective when given prior to the onset of myocardial ischaemia, although their physiological functions are varied and the mechanisms of their cardioprotective actions appear to be diverse and often ill defined. However, one common denominator that is emerging is the ability of these peptides to modulate mast cell degranulation, raising the possibility that peptide-induced mast cell degranulation or stabilization may hold the key to a common mechanism of their cardioprotection. 3 The aim of this review was to consolidate the evidence implying that mast cell degranulation could play both a detrimental and protective role in myocardial ischaemia, depending upon when it occurs, and that this may underlie the cardioprotective effects of a range of diverse peptides that exerts physiological effects within the cardiovascular system.

  7. Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier.

    Science.gov (United States)

    Ruan, Renquan; Chen, Ming; Sun, Sijie; Wei, Pengfei; Zou, Lili; Liu, Jing; Gao, Dayong; Wen, Longping; Ding, Weiping

    2016-01-01

    Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders. PMID:27374619

  8. Liposome-based engineering of cells to package hydrophobic compounds in membrane vesicles for tumor penetration.

    Science.gov (United States)

    Lee, Junsung; Kim, Jiyoung; Jeong, Moonkyoung; Lee, Hyoungjin; Goh, Unbyeol; Kim, Hyaeyeong; Kim, Byungji; Park, Ji-Ho

    2015-05-13

    Natural membrane vesicles (MVs) derived from various types of cells play an essential role in transporting biological materials between cells. Here, we show that exogenous compounds are packaged in the MVs by engineering the parental cells via liposomes, and the MVs mediate autonomous intercellular migration of the compounds through multiple cancer cell layers. Hydrophobic compounds delivered selectively to the plasma membrane of cancer cells using synthetic membrane fusogenic liposomes were efficiently incorporated into the membrane of MVs secreted from the cells and then transferred to neighboring cells via the MVs. This liposome-mediated MV engineering strategy allowed hydrophobic photosensitizers to significantly penetrate both spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy. These results suggest that innate biological transport systems can be in situ engineered via synthetic liposomes to guide the penetration of chemotherapeutics across challenging tissue barriers in solid tumors.

  9. T cells recognizing a peptide contaminant undetectable by mass spectrometry

    DEFF Research Database (Denmark)

    Brezar, Vedran; Culina, Slobodan; Østerbye, Thomas;

    2011-01-01

    Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility...... complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid...

  10. Effects of antiphagocytic agents on penetration of Eimeria magna sporozoites into cultured cells.

    Science.gov (United States)

    Jensen, J B; Edgar, S A

    1976-04-01

    Madin-Darby Bovine Kidney cells were treated with sodium flouride, iodoacetate, and 2-deosyglucose, reagents that block glycolysis, and thus reduce phagocytosis. Sporozoites readily entered cells whose ATP stores were largely depleted. They also entered cells treated with colchicine, colcemid, and vinblastine. These latter agents did not inhibit sporozite motility after 6 hr incubation. Cytochalasin B prevented penetration of cells by inhibiting the motility of sporozoites. This effect was reversible. Warm sporozoites entered cold cells 4 times more radily than cold sporozoites into warm cells. The above findings suggest that phagocytosis is not the mechanism for entry of E. magna sporozoites into cultured cells, but that sporozoite motility is of primary importance.

  11. Cell-Penetrating Poly(disulfide) Assisted Intracellular Delivery of Mesoporous Silica Nanoparticles for Inhibition of miR-21 Function and Detection of Subsequent Therapeutic Effects.

    Science.gov (United States)

    Yu, Changmin; Qian, Linghui; Ge, Jingyan; Fu, Jiaqi; Yuan, Peiyan; Yao, Samantha C L; Yao, Shao Q

    2016-08-01

    The design of drug delivery systems capable of minimal endolysosomal trapping, controlled drug release, and real-time monitoring of drug effect is highly desirable for personalized medicine. Herein, by using mesoporous silica nanoparticles (MSNs) coated with cell-penetrating poly(disulfide)s and a fluorogenic apoptosis-detecting peptide (DEVD-AAN), we have developed a platform that could be uptaken rapidly by mammalian cells via endocytosis-independent pathways. Subsequent loading of these MSNs with small molecule inhibitors and antisense oligonucleotides resulted in intracellular release of these drugs, leading to combination inhibition of endogenous miR-21 activities which was immediately detectable by the MSN surface-coated peptide using two-photon fluorescence microscopy. PMID:27325284

  12. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B

    2014-07-01

    Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

  13. Binding and Clustering of Glycosaminoglycans: A Common Property of Mono- and Multivalent Cell-Penetrating Compounds

    OpenAIRE

    Ziegler, André; Seelig, Joachim

    2007-01-01

    Recent observations in cell culture provide evidence that negatively charged glycosaminoglycans (GAGs) at the surface of biological cells bind cationic cell-penetrating compounds (CPCs) and cluster during CPC binding, thereby contributing to their endocytotic uptake. The GAG binding and clustering occur in the low-micromolar concentration range and suggest a tight interaction between GAGs and CPCs, although the relation between binding affinity and specificity of this interaction remains to b...

  14. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E;

    2012-01-01

    Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...... have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA...

  15. T Cell Epitope Peptide Therapy for Allergic Diseases.

    Science.gov (United States)

    O'Hehir, Robyn E; Prickett, Sara R; Rolland, Jennifer M

    2016-02-01

    Careful selection of dominant T cell epitope peptides of major allergens that display degeneracy for binding to a wide array of MHC class II molecules allows induction of clinical and immunological tolerance to allergen in a refined treatment strategy. From the original concept of peptide-induced T cell anergy arising from in vitro studies, proof-of-concept murine models and flourishing human trials followed. Current randomized, double-blind, placebo-controlled clinical trials of mixtures of T cell-reactive short allergen peptides or long contiguous overlapping peptides are encouraging with intradermal administration into non-inflamed skin a preferred delivery. Definitive immunological mechanisms are yet to be resolved but specific anergy, Th2 cell deletion, immune deviation, and Treg induction seem implicated. Significant efficacy, particularly with short treatment courses, in a range of aeroallergen therapies (cat, house dust mite, grass pollen) with inconsequential non-systemic adverse events likely heralds a new class of therapeutic for allergy, Synthetic Peptide Immuno-Regulatory Epitopes (SPIRE). PMID:26768622

  16. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans

    KAUST Repository

    Åmand, Helene L.

    2012-11-01

    Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide\\'s ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell. © 2012 Elsevier B.V.

  17. A peptide-based fluorescent chemosensor for measuring cadmium ions in aqueous solutions and live cells.

    Science.gov (United States)

    Wang, Peng; Wu, Jiang; Liu, Lixuan; Zhou, Panpan; Ge, Yushu; Liu, Dan; Liu, Weisheng; Tang, Yu

    2015-11-01

    A novel peptide fluorescent chemosensor (H2L) with a lysine backbone and both -NH2 sites conjugated with cysteine and dansyl groups has been designed and synthesized by solid phase peptide synthesis with Fmoc chemistry. This chemosensor is a promising analytical tool for detecting Cd(2+) based on the photo-induced electron transfer (PET) effect by turn-on response in 100% aqueous solutions. As designed, H2L exhibits excellent cell permeation and low biotoxicity as well as displaying relatively high selectivity and sensitivity. The chemosensor penetrated live HeLa cells and detected intracellular Cd(2+) by turn-on response. The binding stoichiometry and affinity, interference test, pH sensitivity, fluorescence quantum yield, quantum mechanical calculations, lifetimes, and cytotoxicity of the chemosensor H2L to Cd(2+) were also investigated. Moreover, H2L exhibits low biotoxicity with a limit of detection (LOD) for Cd(2+) of about 52 nM, implying that H2L can be used as a highly selective and sensitive peptide fluorescent chemosensor in biological systems. PMID:26411376

  18. The Antitumor Peptide CIGB-552 Increases COMMD1 and Inhibits Growth of Human Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Julio R. Fernández Massó

    2013-01-01

    Full Text Available We have demonstrated that the peptide L-2 designed from an alanine scanning of the Limulus-derived LALF32-51 region is a potential candidate for the anticancer therapy and its cell-penetrating capacity is an associated useful property. By the modification in the primary structure of L-2, a second-generation peptide (CIGB-552 was developed. However, the molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-κB signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-κB, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 decreases the antioxidant capacity and induces the peroxidation of proteins and lipids in the tumor cells. Altogether, this study provides new insights into the mechanism of action of the peptide CIGB-552, which could be relevant in the design of future anticancer therapies.

  19. A novel peptide, selected from phage display library of random peptides, can efficiently target into human breast cancer cell

    Institute of Scientific and Technical Information of China (English)

    DONG Jian; LIU WeiQing; JIANG AiMei; ZHANG KeJian; CHEN MingQing

    2008-01-01

    To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex-tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep-tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur-ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca-pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.

  20. Cell Penetrable Human scFv Specific to Middle Domain of Matrix Protein-1 Protects Mice from Lethal Influenza

    Directory of Open Access Journals (Sweden)

    Fonthip Dong-din-on

    2015-01-01

    Full Text Available A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1 is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN. The PEN-scFv (transbody, when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

  1. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

    OpenAIRE

    Yin, HaiFang; Boisguerin, Prisca; Moulton, Hong M.; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew JA

    2013-01-01

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was ...

  2. Effects of HepⅡ domain peptides Ⅴ of fibronectin on corneal permeability, endothelial cells, intraocular pressure and morphology of trabecular meshwork in rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qing-wei; WANG Ning-li; LIU Xu-yang; CHEN Feng-hua; LI Ping-yu; WANG Xiao-zhen

    2011-01-01

    Background Trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow in the eye. This study aimed to evaluate the role of HepⅡ domain peptides Ⅴ on corneal permeability, corneal endothelial cells, intraocular pressure (IOP) and morphology of trabecular meshwork in rats.Methods The IOP of rat eyes was measured before and 3, 5, 7 and 8 hours after topical delivery of HepⅡ domain peptides Ⅴ through intracameral injections. The peptide's concentration in aqueous humor was assessed by high performance liquid chromatography (HPLC). The shape and density of endothelial cells were observed by laser confocal microscopy 8 hours, 3 and 14 days after intracameral injections of HepⅡ domain peptides Ⅴ. The morphological changes in TM of rat eyes were assessed by transmission electron microscopy (TEM).Results Intracameral injection of HepⅡ domain peptides Ⅴ significantly (P <0.001) decreased IOP by (5.71 ±2.10) mmHg in rats at 5 hours after injection. There were no obvious changes of the shape and the density of corneal endothelial cells. In addition, morphological changes in the TM of rats were observed including the expansion of intercellular spaces in the juxtacanalicular meshwork, removal of extracellular material, cellular relaxation, and cytoskeleton reorganization.Conclusions HepⅡ domain peptides Ⅴ could not penetrate cornea and was safe to corneal endothelial cells. HepⅡ domain peptides Ⅴ could significantly decrease IOP in rat probably by disorganizing actin cytoskeleton and cell-junction in the TM.

  3. Integration of antimicrobial peptides with gold nanoparticles as unique non-viral vectors for gene delivery to mesenchymal stem cells with antibacterial activity.

    Science.gov (United States)

    Peng, Li-Hua; Huang, Yan-Fen; Zhang, Chen-Zhen; Niu, Jie; Chen, Ying; Chu, Yang; Jiang, Zhi-Hong; Gao, Jian-Qing; Mao, Zheng-Wei

    2016-10-01

    Gold nanoparticles (AuNPs) have emerged as attractive non-viral gene vectors. However their application in regenerative medicine is still limited partially due to a lack of an intrinsic capacity to transfect difficult-to-transfect cells such as primary cells or stem cells. In current study, we report the synthesis of antimicrobial peptide conjugated cationic AuNPs (AuNPs@PEP) as highly efficient carriers for gene delivery to stem cells with antibacterial ability. The AuNPs@PEP integrate the advantages of cationic AuNPs and antibacterial peptides: the presence of cationic AuNPs can effectively condense DNA and the antimicrobial peptides are essential for the cellular & nucleus entry enhancement to achieve high transfection efficiency and antibacterial ability. As a result, antimicrobial peptides conjugated AuNPs significantly promoted the gene transfection efficiency in rat mesenchymal stem cells than pristine AuNPs, with a similar extent to those expressed by TAT (a well-known cell-penetrating peptide) modified AuNPs. More interestingly, the combinational system has better antibacterial ability than free antimicrobial peptides in vitro and in vivo, possibly due to the high density of peptides on the surface of AuNPs. Finally we present the concept-proving results that AuPs@PEP can be used as a carrier for in vivo gene activation in tissue regeneration, suggesting its potential as a multifunctional system with both gene delivery and antibacterial abilities in clinic. PMID:27376562

  4. The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration.

    Science.gov (United States)

    Xu, Jingwei; Gui, Miao; Wang, Dianhong; Xiang, Ye

    2016-06-23

    Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Å crystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane. PMID:27309813

  5. Micro-structured peptide surfaces for the detection of high-affinity peptide-receptor interactions in living cells.

    Science.gov (United States)

    Lipp, Anna-Maria; Ji, Bozhi; Hager, Roland; Haas, Sandra; Schweiggl, Simone; Sonnleitner, Alois; Haselgrübler, Thomas

    2015-12-15

    Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged β3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment. PMID:26210593

  6. Protease-activatable organometal-Peptide bioconjugates with enhanced cytotoxicity on cancer cells.

    Science.gov (United States)

    Splith, Katrin; Hu, Wanning; Schatzschneider, Ulrich; Gust, Ronald; Ott, Ingo; Onambele, Liliane A; Prokop, Aram; Neundorf, Ines

    2010-07-21

    Over the past years, numerous promising new metalorganic lead structures have been developed exhibiting highly active cytostatic properties. However, the efficiency of such chemotherapeutics in the treatment of tumors is often limited by their low therapeutic index due to their short half-life, lack of tumor selectivity, and associated side effects. Furthermore, the membrane barrier often restricts their cellular uptake by passive diffusion. In this contribution, we describe the synthesis, cellular uptake, and biologic activity of a series of cymantrene-peptide conjugates. Cymantrene CpMn(CO)(3) is a robust organometallic group, which is stable in air and water and easy to functionalize. In this work, some new cymantrene derivatives with different linkers between the half-sandwich complex and the carboxylate group were attached to the cell-penetrating peptide sC18 that should act as a transporter for the metal moiety. All conjugates were characterized for their cytotoxic activity on human breast adenocarcinoma cells (MCF-7) and human colon carcinoma cells (HT-29). We found that bioconjugates bearing two cymantrene groups were more active than the monofunctionalized ones. By the introduction of a cathepsin B cleavage site next to the organometallic group, the biologic activity could be in increased even further. Fluorescence microscopy studies and apoptosis assays gave preliminary hints on the mode of action of these systems. PMID:20586419

  7. Intracellular targets of RGDS peptide in melanoma cells

    Directory of Open Access Journals (Sweden)

    Capogrossi Maurizio C

    2010-04-01

    Full Text Available Abstract Background RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment. Results In the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 μM and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA. Conclusions RGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular

  8. Peptide imprinted receptors for the determination of the small cell lung cancer associated biomarker progastrin releasing peptide

    DEFF Research Database (Denmark)

    Qader, A. A.; Urraca, J.; Torsetnes, S. B.;

    2014-01-01

    prior to LCMS based quantification. Peptide imprinted polymers with the best affinity characteristics were first identified from a 96-polymer combinatorial library. The effects of functional monomers, crosslinker, porogen, and template on adsorption capacity and selectivity for NLLGLIEAK were......Peptide imprinted polymers were developed for detection of progastrin releasing peptide (ProGRP); a low abundant blood based biomarker for small cell lung cancer. The polymers targeted the proteotypic nona-peptide sequence NLLGLIEAK and were used for selective enrichment of the proteotypic peptide...... investigated and optimized. Ultimately, a solid phase extraction method was developed for highly selective enrichment of the target peptide from tryptic digests. (C) 2014 Elsevier B.V. All rights reserved....

  9. Vasoactive intestinal peptide (VIP) inhibits human renal cell carcinoma proliferation.

    Science.gov (United States)

    Vacas, Eva; Fernández-Martínez, Ana B; Bajo, Ana M; Sánchez-Chapado, Manuel; Schally, Andrew V; Prieto, Juan C; Carmena, María J

    2012-10-01

    Clear renal cell carcinoma (cRCC) is an aggressive and fatal neoplasm. The present work was undertaken to investigate the antiproliferative potential of vasoactive intestinal peptide (VIP) exposure on non-tumoral (HK2) and tumoral (A498, cRCC) human proximal tubular epithelial cell lines. Reverse transcription and semiquantitative PCR was used at the VIP mRNA level whereas enzyme immunoanalysis was performed at the protein level. Both renal cell lines expressed VIP as well as VIP/pituitary adenylate cyclase-activating peptide (VPAC) receptors whereas only HK2 cells expressed formyl peptide receptor-like 1 (FPRL-1). Receptors were functional, as shown by VIP stimulation of adenylyl cyclase activity. Treatment with 0.1μM VIP (24h) inhibited proliferation of A498 but not HK2 cells as based on a reduction in the incorporation of [(3)H]-thymidine and BrdU (5'-Br-2'-deoxyuridine), PCNA (proliferating-cell nuclear antigen) expression and STAT3 (signal transducer and activator of transcription 3) expression and activation. VPAC(1)-receptor participation was established using JV-1-53 antagonist and siRNA transfection. Growth-inhibitory response to VIP was related to the cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP (EPAC)/phosphoinositide 3-kinase (PI3-K) signaling systems as shown by studies on adenylate cyclase stimulation, and using the EPAC-specific compound 8CPT-2Me-cAMP and specific kinase inhibitors such as H89, wortmannin and PD98059. The efficacy of VIP on the prevention of tumor progression was confirmed in vivo using xenografted athymic mouse. These actions support a potential role of this peptide and its agonists in new therapies for cRCC.

  10. Parametric analysis of neutron streaming through major penetrations in the 0.914 m TFTR test cell floor

    International Nuclear Information System (INIS)

    Neutron streaming through penetrations in the 0.914 m TFTR test cell floor has two distinct features: (1) the oblique angle of incidence; and (2) the high order of anisotropy in the angular distribution for incident neutrons with energies > 10 keV. The effects of these features on the neutron streaming into the TFTR basement were studied parametrically for isolated penetrations. Variations with respect to the source energies, angular distributions, and sizes of the penetrations were made. The results form a data base from which the spatial distribution of the neutron flux in the basement due to multiple penetrations may be evaluated

  11. IDENTIFICATION OF SPECIFIC PEPTIDE LIGANDS FOR B-LYMPHOMA CELL AND ITS EFFECT ON TYROSINE PHOSPHORYLATION AND CELL APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    宋良文; 马宪梅; 崔雪梅; 李扬; 王晓民

    2004-01-01

    Objective To search novel method for diagnosis and therapy of B-lymphoma, specific small molecular peptide ligands against binding site of tumor cells were screened and its effects on signal transduction and cell apoptosis were tested. Methods Specific peptide ligands were screened by binding with site of human B lymphoma cell (OC1LY8) using peptide-bead libraries. The identified peptides were characterized with responsible cells by rebinding test. The role of tyrosine phosphorylation of peptide ligand was tested by Western blot;and its apoptosispromoting role was observed by confocal fluorescent microscope. Results Specific peptide ligand was able to bind specifically to site on cell surface and enter into cytoplasm. Tetrameric peptide ligand was able to strongly trigger signal transduction resulting in tyrosine phosphorylation and cellular apoptosis in OC1LY8 cell line.Conclusion Screened peptide ligand can effectively bind with OC1LY8 cell, stimulate cellular tyrosine phosphorylation and induce cellular apoptosis.

  12. Tumor-Homing and Penetrating Peptide-Functionalized Photosensitizer-Conjugated PEG-PLA Nanoparticles for Chemo-Photodynamic Combination Therapy of Drug-Resistant Cancer.

    Science.gov (United States)

    Feng, Xingye; Jiang, Di; Kang, Ting; Yao, Jianhui; Jing, Yixian; Jiang, Tianze; Feng, Jingxian; Zhu, Qianqian; Song, Qingxiang; Dong, Nan; Gao, Xiaoling; Chen, Jun

    2016-07-20

    The combination of photodynamic therapy (PDT) and chemotherapy holds great potential in combating drug-resistant cancers. However, the major challenge that lies ahead is how to achieve high coloading capacity for both photosensitizer and chemo-drugs and how to gain efficient delivery of drugs to the drug-resistant tumors. In this study, we prepared a nanovehicle for codelivery of photosensitizer (pyropheophorbide-a, PPa) and chemo-drugs (paclitaxel, PTX) based on the synthesis of PPa-conjugated amphiphilic copolymer PPa-PLA-PEG-PLA-PPa. The obtained nanoparticles (PP NP) exhibited a satisfactory high drug-loading capacity for both drugs. To achieve effective tumor-targeting therapy, the surface of PP NP was decorated with a tumor-homing and penetrating peptide F3. In vitro cellular experiments showed that F3-functionalized PP NP (F3-PP NP) exhibited higher cellular association than PP NP and resulted in the strongest antiproliferation effect. In addition, compared with the unmodified nanoparticles, F3-PP NP exhibited a more preferential enrichment at the tumor site. Pharmacodynamics evaluation in vivo demonstrated that a longer survival time was achieved by the tumor-bearing mice treated with PP NP (+laser) than those treated with chemotherapy only or PDT only. Such antitumor efficacy of combination therapy was further improved following the F3 peptide functionalization. Collectively, these results suggested that targeted combination therapy may pave a promising way for the therapy of drug-resistant tumor. PMID:27332148

  13. Peptides that influence membrane topology

    Science.gov (United States)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  14. Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Park YJ

    2012-09-01

    Full Text Available Yoon Jung Choi,1,* Jue Yeon Lee,2,* Chong Pyoung Chung,2 Yoon Jeong Park,1,21Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Human dental pulp stem cells (DPSCs have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1 was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP, and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.Keywords: superoxide

  15. Adhesion between peptides/antibodies and breast cancer cells

    Science.gov (United States)

    Meng, J.; Paetzell, E.; Bogorad, A.; Soboyejo, W. O.

    2010-06-01

    Atomic force microscopy (AFM) techniques were used to measure the adhesion forces between the receptors on breast cancer cells specific to human luteinizing hormone-releasing hormone (LHRH) peptides and antibodies specific to the EphA2 receptor. The adhesion forces between LHRH-coated AFM tips and human MDA-MB-231 cells (breast cancer cells) were shown to be about five times greater than those between LHRH-coated AFM tips and normal Hs578Bst breast cells. Similarly, those between EphA2 antibody-coated AFM tips and breast cancer cells were over five times greater than those between EphA2 antibody-coated AFM tips and normal breast cells. The results suggest that AFM can be used for the detection of breast cancer cells in biopsies. The implications of the results are also discussed for the early detection and localized treatment of cancer.

  16. Penetration of fosfomycin into IPEC-J2 cells in the presence or absence of deoxynivalenol.

    Directory of Open Access Journals (Sweden)

    Guadalupe Martínez

    Full Text Available Fosfomycin (FOS is an antibiotic used in pig farms for treatment and prevention of infections caused by resistant bacteria during the post-weaning period. Antibiotics and non-toxic concentrations of mycotoxins, such as deoxynivalenol (DON are frequently found in the diet of animals. These compounds can establish interactions in the intestinal tract, which could affect and/or modify the penetration of FOS to enterocytes. The aim of this study was to determine the penetration of FOS into IPECJ-2 cells, a cell line derived from the small intestine of piglets, in the presence and absence of DON. The results from this study showed that there was statistically significant difference in the intracellular concentration of FOS between cells incubated with 580 µg/ml FOS and cells incubated with 580 µg/ml FOS and 1 µg/ml DON. The Cmax of the intracellular antibiotic in the culture plates incubated with FOS in absence of DON was 45.81 µg/ml with a tmax of 4 h. When IPEC-2 cells were incubated with FOS and DON the Cmax was 20.06 µg/ml and the tmax was 30 min. It is concluded that the non-toxic concentration of DON on IPEC-J2 cells after short-term exposure, interferes with the pharmacokinetics of the antibiotic.

  17. Peptide-templating dye-sensitized solar cells

    Science.gov (United States)

    Han, Tae Hee; Moon, Hyoung-Seok; Hwang, Jin Ok; Seok, Sang Il; Im, Sang Hyuk; Ouk Kim, Sang

    2010-05-01

    A hollow TiO2 nanoribbon network electrode for dye-sensitized solar cells (DSSC) was fabricated by a biotemplating process combining peptide self-assembly and atomic layer deposition (ALD). An aromatic peptide of diphenylalanine was assembled into a three-dimensional network consisting of highly entangled nanoribbons. A thin TiO2 layer was deposited at the surface of the peptide template via the ALD process. After the pyrolysis of the peptide template, a highly entangled nanotubular TiO2 framework was successfully prepared. Evolution of the crystal phase and crystallite size of the TiO2 nanostructure was exploited by controlling the calcination temperature. Finally, the hollow TiO2 nanoribbon network electrode was integrated into DSSC devices and their photochemical performances were investigated. Hollow TiO2 nanoribbon-based DSSCs exhibited a power conversion efficiency of 3.8%, which is comparable to the conventional TiO2 nanoparticle-based DSSCs (3.5%). Our approach offers a novel pathway for DSSCs consisting of TiO2 electrodes via biotemplating.

  18. Peptide-templating dye-sensitized solar cells

    International Nuclear Information System (INIS)

    A hollow TiO2 nanoribbon network electrode for dye-sensitized solar cells (DSSC) was fabricated by a biotemplating process combining peptide self-assembly and atomic layer deposition (ALD). An aromatic peptide of diphenylalanine was assembled into a three-dimensional network consisting of highly entangled nanoribbons. A thin TiO2 layer was deposited at the surface of the peptide template via the ALD process. After the pyrolysis of the peptide template, a highly entangled nanotubular TiO2 framework was successfully prepared. Evolution of the crystal phase and crystallite size of the TiO2 nanostructure was exploited by controlling the calcination temperature. Finally, the hollow TiO2 nanoribbon network electrode was integrated into DSSC devices and their photochemical performances were investigated. Hollow TiO2 nanoribbon-based DSSCs exhibited a power conversion efficiency of 3.8%, which is comparable to the conventional TiO2 nanoparticle-based DSSCs (3.5%). Our approach offers a novel pathway for DSSCs consisting of TiO2 electrodes via biotemplating.

  19. Peptide-templating dye-sensitized solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Tae Hee; Moon, Hyoung-Seok; Hwang, Jin Ok; Kim, Sang Ouk [Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701 (Korea, Republic of); Seok, Sang Il; Im, Sang Hyuk, E-mail: imromy@krict.re.kr, E-mail: sangouk.kim@kaist.ac.kr [KRICT-EPFL Global Research Laboratory, Advanced Materials Division, Korea Research Institute of Chemical Technology, Daejeon 305-600 (Korea, Republic of)

    2010-05-07

    A hollow TiO{sub 2} nanoribbon network electrode for dye-sensitized solar cells (DSSC) was fabricated by a biotemplating process combining peptide self-assembly and atomic layer deposition (ALD). An aromatic peptide of diphenylalanine was assembled into a three-dimensional network consisting of highly entangled nanoribbons. A thin TiO{sub 2} layer was deposited at the surface of the peptide template via the ALD process. After the pyrolysis of the peptide template, a highly entangled nanotubular TiO{sub 2} framework was successfully prepared. Evolution of the crystal phase and crystallite size of the TiO{sub 2} nanostructure was exploited by controlling the calcination temperature. Finally, the hollow TiO{sub 2} nanoribbon network electrode was integrated into DSSC devices and their photochemical performances were investigated. Hollow TiO{sub 2} nanoribbon-based DSSCs exhibited a power conversion efficiency of 3.8%, which is comparable to the conventional TiO{sub 2} nanoparticle-based DSSCs (3.5%). Our approach offers a novel pathway for DSSCs consisting of TiO{sub 2} electrodes via biotemplating.

  20. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.

    Science.gov (United States)

    Hayashi, Moyuru; Tamura, Yosuke; Dohmae, Naoshi; Kojima, Soichi; Shimonaka, Motoyuki

    2008-05-01

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides. PMID:18294956

  1. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

    International Nuclear Information System (INIS)

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides

  2. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  3. Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.

    Science.gov (United States)

    Park, Hee Ho; Sohn, Youngsoo; Yeo, Ji Woo; Park, Ju Hyun; Lee, Hong Jai; Ryu, Jina; Rhee, Won Jong; Park, Tai Hyun

    2014-12-01

    Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

  4. Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

    Directory of Open Access Journals (Sweden)

    Lösche Mathias

    2011-09-01

    Full Text Available Abstract Background Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments. Results SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles. Conclusions These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.

  5. Requirements for Peptide-induced T Cell Receptor Downregulation on Naive CD8+ T Cells

    OpenAIRE

    Cai, Zeling; Kishimoto, Hidehiro; Brunmark, Anders; Jackson, Michael R.; Peterson, Per A.; Sprent, Jonathan

    1997-01-01

    The requirements for inducing downregulation of α/β T cell receptor (TCR) molecules on naive major histocompatibility complex class I–restricted T cells was investigated with 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required CD8 expression on the responder cells and was heavily dependent upon costimulation provided by either B7-1 or ICAM-1 on antigen-presenting cells (APC). These stringent re...

  6. Enhanced metalloadsorption of bacterial cells displaying poly-His peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sousa, C.; Cebolla, A.; Lorenzo, V. de [CSIC, Madrid (Spain)

    1996-08-01

    The properties of Escherichia coli cells, acquired by cell surface presentation of one or two hexahistidine (His) clusters carried by the outer membrane LamB protein, have been examined. Strains producing LamB hybrids with the His chains accumulated greater than 11-fold more Cd{sup 2} than E. coli cells expressing the protein without the His insert. Furthermore, the hexa-His chains on the cell surface caused cells to adhere reversibly to a Ni{sup 2+}-containing solid matrix in a metal-dependent fashion. Thus, expression of poly-His peptides enables bacteria to act as a metalloaffinity adsorbent. These results open up the possibility for biosorption of heavy ions using engineered microorganisms. 32 refs., 3 figs.

  7. Biokinetics and dosimetry of several radiolabelled peptides in cancer cells

    Science.gov (United States)

    Rodríguez-Cortés, J.; Ferro-Flores, G.; de Murphy, C. Arteaga; Pedraza-López, M.; Ramírez-Iglesias, M. A. T.

    Radiolabelled peptides have been used as target-specific radiopharmaceuticals. The goal of this research was the in vitro assessment of the uptake, internalization, externalization, and efflux of five radiolabelled peptides in cancer cells to estimate radiation-absorbed doses from experimental biokinetic data. 177Lu-DOTA-octreotate, 188Re-lanreotide, and 99mTc-HYNIC-octreotide were studied in the AR42J cell line. The PC3 and NCIH69 cells were used for 99mTc-HYNIC-bombesin and 177Lu-DOTA-minigastrin, respectively. The cumulated activities in the membrane and cytoplasm were calculated by integration of the experimental time-activity curves and used for dosimetry calculations according to the Medical Internal Radiation Dose (MIRD) cellular methodology. The mean absorbed dose to the cell nucleus were 0.69±0.09, 0.11±0.08, 0.55±0.09, 3.45±0.48, and 3.30±0.65 Gy/Bq for 99mTc-HYNIC-bombesin, 99mTc-HYNIC-octreotide, 177Lu-DOTA-minigastrin, 177Lu-DOTA-octreotate, and 188Re-lanreotide, respectively. If radiopharmaceutical cell kinetics were not used and only uptake data were considered, the calculated doses would be overestimated up to 25 times.

  8. Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization

    Energy Technology Data Exchange (ETDEWEB)

    Batenburg, A.M.; Demel, R.A.; Verkleij, A.J.; de Kruijff, B.

    1988-07-26

    In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary /sup 31/P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.

  9. Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization

    International Nuclear Information System (INIS)

    In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure

  10. p53 Aggregates penetrate cells and induce the co-aggregation of intracellular p53.

    Directory of Open Access Journals (Sweden)

    Karolyn J Forget

    Full Text Available Prion diseases are unique pathologies in which the infectious particles are prions, a protein aggregate. The prion protein has many particular features, such as spontaneous aggregation, conformation transmission to other native PrP proteins and transmission from an individual to another. Protein aggregation is now frequently associated to many human diseases, for example Alzheimer's disease, Parkinson's disease or type 2 diabetes. A few proteins associated to these conformational diseases are part of a new category of proteins, called prionoids: proteins that share some, but not all, of the characteristics associated with prions. The p53 protein, a transcription factor that plays a major role in cancer, has recently been suggested to be a possible prionoid. The protein has been shown to accumulate in multiple cancer cell types, and its aggregation has also been reproduced in vitro by many independent groups. These observations suggest a role for p53 aggregates in cancer development. This study aims to test the «prion-like» features of p53. Our results show in vitro aggregation of the full length and N-terminally truncated protein (p53C, and penetration of these aggregates into cells. According to our findings, the aggregates enter cells using macropinocytosis, a non-specific pathway of entry. Lastly, we also show that once internalized by the cell, p53C aggregates can co-aggregate with endogenous p53 protein. Together, these findings suggest prion-like characteristics for p53 protein, based on the fact that p53 can spontaneously aggregate, these aggregates can penetrate cells and co-aggregate with cellular p53.

  11. Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation

    Directory of Open Access Journals (Sweden)

    Kang MJ

    2013-03-01

    Full Text Available Myung Joo Kang, Sang Han Park, Mean Hyung Kang, Min Jung Park, Young Wook Choi College of Pharmacy, Chung-Ang University, Seoul, Korea Background: A novel dual ligand–modified liposome, folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier (FP-Lipo, was designed to overcome the nonselectivity of conventional penetrating peptide-tagged nanoparticulates and to provide the advantage of selective targeting of the folic acid receptor, which is frequently overexpressed on epithelial cancer cells. Methods: FP-Lipo was prepared by a sequential process of formation of a maleimide-derivatized small unilamellar vesicle, postinsertion of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000–folate to the vesicle, and Pep-1 peptide conjugation via thiol-maleimide linkage. Conformational and physical characteristics of the FP-Lipo nanocarriers were investigated for the extent of Pep-1 peptide and folic acid on the surface, vesicle size, and zeta potential. In vitro cellular uptake behaviors of the novel carrier containing a fluorescein dextran isothiocyanate probe were examined by spectrophotometry or by confocal laser scanning microscopy. Results: A novel nanocarrier bearing approximately 750 folate ligands and 100 penetrating peptides per vesicle was successfully prepared. The physical properties were as follows: 140 nm in size; 5 mV in zeta potential; less than 0.3 in polydispersity index. An in vitro cellular uptake study revealed that the FP-Lipo nanocarrier system exhibited more than twofold enhanced translocation into the folic acid receptor–positive HeLa cells compared with the single Pep-1 peptide–modified liposome. Meanwhile, its cellular association and internalization into the folic acid receptor–negative normal HaCaT cells was comparable with that of Pep-1 peptide–modified liposome. Conclusion: An advanced dual ligand-modified liposome is potentially useful for the treatment of folic acid receptor

  12. Differential regulation of cell functions by CSD peptide subdomains

    OpenAIRE

    Reese, Charles; Dyer, Shanice; Perry, Beth; Bonner, Michael; Oates, James; Hofbauer, Ann; Sessa, William; Bernatchez, Pascal; Visconti, Richard P; Zhang, Jing; Hatfield, Corey M; Silver, Richard M.; Hoffman, Stanley; Tourkina, Elena

    2013-01-01

    Background In fibrotic lung diseases, expression of caveolin-1 is decreased in fibroblasts and monocytes. The effects of this deficiency are reversed by treating cells or animals with the caveolin-1 scaffolding domain peptide (CSD, amino acids 82–101 of caveolin-1) which compensates for the lack of caveolin-1. Here we compare the function of CSD subdomains (Cav-A, Cav-B, Cav-C, Cav-AB, and Cav-BC) and mutated versions of CSD (F92A and T90A/T91A/F92A). Methods Migration toward the chemokine CX...

  13. Self-Assembled Peptide Gels for 3D Cell Culture

    OpenAIRE

    Tang, Claire

    2010-01-01

    Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progre...

  14. Selenium as an alternative peptide label - comparison to fluorophore-labelled penetratin

    DEFF Research Database (Denmark)

    Hyrup Møller, Laura; Bahnsen, Jesper Søborg; Nielsen, Hanne Mørck;

    2015-01-01

    In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenMSe, TAMRA...

  15. A PCNA-derived cell permeable peptide selectively inhibits neuroblastoma cell growth.

    Directory of Open Access Journals (Sweden)

    Long Gu

    Full Text Available Proliferating cell nuclear antigen (PCNA, through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA, which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.

  16. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles.

    Science.gov (United States)

    Torrano, Adriano A; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ∼50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ∼150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine. PMID:27341699

  17. Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

    Directory of Open Access Journals (Sweden)

    Lindin Inger

    2011-03-01

    Full Text Available Abstract Background Several naturally occurring cationic antimicrobial peptides (CAPs, including bovine lactoferricin (LfcinB, display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS.

  18. Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

    Science.gov (United States)

    Nojiri, Takashi; Hosoda, Hiroshi; Tokudome, Takeshi; Miura, Koichi; Ishikane, Shin; Otani, Kentaro; Kishimoto, Ichiro; Shintani, Yasushi; Inoue, Masayoshi; Kimura, Toru; Sawabata, Noriyoshi; Minami, Masato; Nakagiri, Tomoyuki; Funaki, Soichiro; Takeuchi, Yukiyasu; Maeda, Hajime; Kidoya, Hiroyasu; Kiyonari, Hiroshi; Shioi, Go; Arai, Yuji; Hasegawa, Takeshi; Takakura, Nobuyuki; Hori, Megumi; Ohno, Yuko; Miyazato, Mikiya; Mochizuki, Naoki; Okumura, Meinoshin; Kangawa, Kenji

    2015-01-01

    Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells. PMID:25775533

  19. Targeting DNA vaccines to myeloid cells using a small peptide.

    Science.gov (United States)

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N

    2015-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

  20. INVESTIGATION OF INDUCING EFFECT OF SPECIFIC CYTOTOXICITY OF CTLS BY ANTIGEN PEPTIDES FROM T LYMPHOCYTIC LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    张桂梅; 黄波; 李东; 王洪涛; 冯作化

    2003-01-01

    Objective: To investigate the characteristics of specific antitumor immunity induced by antigen peptides mixture from T lymphocytic leukemia cells. Method: Antigen peptides mixtures were prepared from different leukemia cell lines and then bound with Hsp70 in vitro. Human peripheral blood mononuclear cells (PBMC) were cultured in vitro, and activated with Hsp70-antigen peptides. The activated PBMC was cultured continuously in vitro, and used as effector cells in vitro test of cytotoxicity to different target cells. Results: The antigen peptides from different leukemia cell lines were peptides mixture and could activate PBMC effectively if they were presented by Hsp70. The activated PBMC could proliferate in the presence of IL-2 and Hsp70-antigen peptides. The proliferative PBMC had specific cytotoxicity to leukemia cells corresponding to the antigen peptides. PBMC activated by antigen peptides from T lymphocytic leukemia cell lines could effectively kill T lymphocytic leukemia cells, and the cytotoxicity of these PBMC to T lymphocytic leukemia cells was significantly stronger than that of PBMC activated by antigen peptides from other leukemia cells (P < 0.05). PBMC activated by either Hut78-peptides or Molt 4-peptides could effectively kill Jurkat cells. And the cytotoxicity of PBMC activated by Hut78/Molt-4-peptides to Jurkat cells was significantly stronger than that of PBMC activated by either Hut78-peptides or Molt-4-peptides alone (P<0.05).Conclusion: Antigen peptides mixture from T lymphocytic leukemia cell lines can induce specific cytotoxic effect to T lymphocytic leukemia cells. There exists cross-reactivity among antigen peptides mixture from different T lymphocytic leukemia cell lines. The cross-reactivity could be amplified by blending of different antigen peptides from different T lymphocytic leukemia cell lines, suggesting that it is possible to prepare broad-spectrum antigen peptide vaccine against T lymphocytic leukemia by using multiple leukemia

  1. Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic...

  2. Somatostatin immunoreactive cells in lesional psoriatic human skin during peptide T treatment.

    Science.gov (United States)

    Johansson, O; Hilliges, M; Talme, T; Marcusson, J A; Wetterberg, L

    1994-03-01

    Peptide T has been shown to be an effective treatment in psoriasis. The mechanism through which peptide T works in psoriasis is at present unknown. Furthermore, a clearance of psoriasis has also been registered using the inhibitory peptide somatostatin. These observations all focus on the fact that peptide T, somatostatin, and/or other peptides, might provide a clue to understanding the etiology and pathogenesis of psoriasis. Therefore, the effect of peptide T administration on somatostatin containing cutaneous cell populations was investigated. Ten psoriatic patients were treated with peptide T (D-Ala-peptide T amide; 2 mg/day i.v.) for 28 days. Serial biopsies were obtained from the psoriatic lesions before, once weekly during and 4 weeks after discontinuation of the peptide T treatment. An indirect immunofluorescence procedure was performed using a polyclonal antiserum against somatostatin. Clinically, most of the patients responded successfully to the treatment. Immunohistochemical investigations of the serial biopsies revealed the appearance of extensive changes in the number of dermal somatostatin immunoreactive dendritic cells. We believe that peptide T may stimulate the local synthesis and/or release of somatostatin, or proliferation and/or migration of certain dendritic cell populations in psoriatic lesions during healing. Since the benefits of peptide T treatment of psoriatic patients parallel earlier investigations using somatostatin infusions, it is likely that somatostatin given exogenously or synthesized/released endogenously plays a vital role in inducing the healing process.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7911613

  3. Formulating tumor-homing peptides as regular nanoparticles enhances receptor-mediated cell penetrability

    OpenAIRE

    Xu, Zhikun; Unzueta Elorza, Ugutz; Roldán, Mónica; Mangues, Ramón; Sánchez Chardi, Alejandro; Ferrer Miralles, Neus; Villaverde Corrales, Antonio; Vázquez Gómez, Esther

    2015-01-01

    The authors acknowledge the financial support granted to E.V. (PI12/00327) and R.M. (PI12/01861) from FIS, to E.V. (TV32013-133930) and to R.M. and A.V. (TV32013-132031) from La Marató de TV3 (416/C/2013), to A.V. from MINECO (Grant BIO2013-41019-P) and from the Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (NANOPROTHER and NANOCOMETS projects). We are grateful to the Protein Production Platform (CIBER-BBN-UAB) for protein production and purif...

  4. Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites.

    Science.gov (United States)

    Li, Yinfeng; Yuan, Hongyan; von dem Bussche, Annette; Creighton, Megan; Hurt, Robert H; Kane, Agnes B; Gao, Huajian

    2013-07-23

    Understanding and controlling the interaction of graphene-based materials with cell membranes is key to the development of graphene-enabled biomedical technologies and to the management of graphene health and safety issues. Very little is known about the fundamental behavior of cell membranes exposed to ultrathin 2D synthetic materials. Here we investigate the interactions of graphene and few-layer graphene (FLG) microsheets with three cell types and with model lipid bilayers by combining coarse-grained molecular dynamics (MD), all-atom MD, analytical modeling, confocal fluorescence imaging, and electron microscopic imaging. The imaging experiments show edge-first uptake and complete internalization for a range of FLG samples of 0.5- to 10-μm lateral dimension. In contrast, the simulations show large energy barriers relative to kBT for membrane penetration by model graphene or FLG microsheets of similar size. More detailed simulations resolve this paradox by showing that entry is initiated at corners or asperities that are abundant along the irregular edges of fabricated graphene materials. Local piercing by these sharp protrusions initiates membrane propagation along the extended graphene edge and thus avoids the high energy barrier calculated in simple idealized MD simulations. We propose that this mechanism allows cellular uptake of even large multilayer sheets of micrometer-scale lateral dimension, which is consistent with our multimodal bioimaging results for primary human keratinocytes, human lung epithelial cells, and murine macrophages.

  5. Kinome profiling using peptide arrays in eukaryotic cells.

    Science.gov (United States)

    Parikh, Kaushal; Peppelenbosch, Maikel P; Ritsema, Tita

    2009-01-01

    Over the last 10 years array and mass spectrometry technologies have enabled the determination of the transcriptome and proteome of biological and in particular eukaryotic systems. This information will likely be of significant value to our elucidation of the molecular mechanisms that govern eukaryotic physiology. However, an equally, if not more important goal, is to define those proteins that participate in signalling pathways that ultimately control cell fate. Enzymes that phosphorylate tyrosine, serine, and threonine residues on other proteins play a major role in signalling cascades that determine cell-cycle entry, and survival and differentiation fate in the tissues across the eukaryotic kingdoms. Knowing which signalling pathways are being used in these cells is of critical importance. Traditional genetic and biochemical approaches can certainly provide answers here, but for technical and practical reasons there is typically pursued one gene or pathway at a time. Thus, a more comprehensive approach is needed in order to reveal signalling pathways active in nucleated cells. Towards this end, kinome analysis techniques using peptide arrays have begun to be applied with substantial success in a variety of organisms from all major branches of eukaryotic life, generating descriptions of cellular signalling without a priori assumptions as to possibly effected pathways. The general procedure and analysis methods are very similar disregarding whether the primary source of the material is animal, plant, or fungal of nature and will be described in this chapter. These studies will help us better understand what signalling pathways are critical to controlling eukaryotic cell function.

  6. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles

    Science.gov (United States)

    Torrano, Adriano A.; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ~50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ~150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine.In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and

  7. Combined Cell Culture-Biosensing Platform Using Vertically Aligned Patterned Peptide Nanofibers for Cellular Studies

    DEFF Research Database (Denmark)

    Taskin, Mehmet B.; Sasso, Luigi; Dimaki, Maria;

    2013-01-01

    This Article presents the development of a combined cell culture–biosensing platform using vertically aligned self-assembled peptide nanofibers. Peptide nanofibers were patterned on a microchip containing gold microelectrodes to provide the cells with a 3D environment enabling them to grow...

  8. Endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty versus penetrating keratoplasty in Asian eyes

    Directory of Open Access Journals (Sweden)

    Ang M

    2012-04-01

    Full Text Available Marcus Ang1,2, Jodhbir S Mehta1–4, Arundhati Anshu1,2, Hon Kiat Wong5, Hla M Htoon2, Donald Tan1–31Singapore National Eye Centre, 2Singapore Eye Research Institute, 3Department of Ophthalmology, National University Health Systems, 4Department of Clinical Sciences, Duke-NUS Graduate Medical School, 5Department of Ophthalmology, Tan Tock Seng Hospital, SingaporeBackground: The purpose of this study was to compare endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty (DSAEK and penetrating keratoplasty in Asian eyes.Methods: This was a retrospective study of patients from our prospective Singapore Corneal Transplant Study cohort who received corneal transplantation in 2006–2008. We compared eyes that underwent DSAEK or penetrating keratoplasty for Fuchs’ endothelial dystrophy or pseudophakic and aphakic bullous keratopathy. Clinical data, and donor and recipient characteristics were recorded. Of 241 patients who met our inclusion criteria, 68 underwent DSAEK and 173 underwent penetrating keratoplasty. The main outcome measure was endothelial cell loss at 1 year. Secondary outcome measures were graft survival and visual outcomes at 1-year follow-up.Results: There were no significant differences in baseline characteristics of patients between the treatment groups. Percent endothelial cell loss at 1-year follow-up was greater in penetrating keratoplasty eyes (40.9% ± 2.9% compared with DSAEK eyes (22.4% ± 2.3%; P < 0.001. DSAEK-treated eyes had significantly superior uncorrected visual acuity (mean difference = 0.42 ± 0.0059; P < 0.001 and best spectacle-corrected visual acuity (mean difference = 0.14 ± 0.032; P < 0.001 as compared with penetrating keratoplasty-treated eyes. Penetrating keratoplasty-treated eyes had worse astigmatism as compared with DSAEK-treated eyes (-3.0 ± 2.1 versus -1.7 ± 0.8; P < 0.001. Graft survival at 1 year was comparable in both groups, ie, 66/68 (97.0% DSAEK-treated eyes

  9. CNS Active O-Linked Glycopeptides that Penetrate the BBB

    Science.gov (United States)

    Jones, Evan; Polt, Robin

    2015-06-01

    Naturally occurring glycopeptides and glycoproteins play important roles in biological processes. Glycosylation is one of the most common post-translational modifications in vivo. Glycopeptides are involved in cell signaling and sorting, providing cell surface markers for recognition. From the drug design and synthesis perspective, modification of a peptide through glycosylation results in increased bioavailability and bioactivity of glycopeptides in living systems with negligible toxicity of degradation products. Glycopeptide synthesis can be accomplished through incorporation of a glycosylated amino acid in solid phase peptide synthesis (SPPS) to form the desired peptide, or via incorporation of sugar-amino acid moieties. Additionally, research indicates that glycosylation increases penetration of the blood-brain barrier (BBB) by peptides, which may lead to novel therapeutics for neurological disorders. Recent applications of glycopeptides have focused on the in vivo central nervous system effects after peripheral administration of centrally active peptides modified with various carbohydrates.

  10. CNS Active O-Linked Glycopeptides that Penetrate the BBB

    Directory of Open Access Journals (Sweden)

    Evan M Jones

    2015-06-01

    Full Text Available Naturally occurring glycopeptides and glycoproteins play important roles in biological processes. Glycosylation is one of the most common post-translational modifications in vivo. Glycopeptides are involved in cell signaling and sorting, providing cell surface markers for recognition. From the drug design and synthesis perspective, modification of a peptide through glycosylation results in increased bioavailability and bioactivity of glycopeptides in living systems with negligible toxicity of degradation products. Glycopeptide synthesis can be accomplished through incorporation of a glycosylated amino acid in solid phase peptide synthesis (SPPS to form the desired peptide, or via incorporation of sugar-amino acid moieties. Additionally, research indicates that glycosylation increases penetration of the blood-brain barrier (BBB by peptides, which may lead to novel therapeutics for neurological disorders. Recent applications of glycopeptides have focused on the in vivo central nervous system effects after peripheral administration of centrally active peptides modified with various carbohydrates.

  11. Cell-permeable Tat-NBD peptide attenuates rat pancreatitis and acinus cell inflammation response

    Institute of Scientific and Technical Information of China (English)

    You-Ming Long; Ken Chen; Xue-Jin Liu; Wen-Rui Xie; Hui Wang

    2009-01-01

    AIM: To investigate the effects of Tat-NEMO-binding domain (NBD) peptide on taurocholate-induced pancreatitis and lipopolysaccharide (LPS)-stimulated AR42J acinus cells inflammatory response in rats. METHODS: Sodium taurocholate (5%) was used to induce the pancreatitis model. Forty-eight rats from the taurocholate group aeceuved an intravenous bolus of 13 mg/kg Tat-NBD (wild-type, WT) peptide, Tat-NBD (mutant-type, MT) peptide, NBD peptide or Tat peptide. The pancreatic histopathology was analyzed by hematoxylin staining. LPS was added to the culture medium to stimulate the AR42J cells. For pretreatment, cells were incubated with different peptides for 2 h before LPS stimulation. Expression of IL-1β and TNF-α mRNA was analyzed using a semi-quantitative reverse-transcript polymerase chain reaction (RT-PCR) method. IL-1β and TNF-α protein in culture medium were detected by enzyme linked immunosorbent assay (ELISA). NF-κB DNA-binding in pancreas was examined by electrophoretic mobility shift assays. P65 expression of AR42J was determined by Strept Actividin-Biotin Complex (SABC) method. RESULTS: Pretreatment with Tat-NBD (WT) peptide at a concentration of 13 mg/kg body wt showed beneficial effect in pancreaitis model. LPS (10 mg/L) resulted in an increase of IL-1β mRNA, IL-1β protein, TNF-α mRNA and TNF-α protein, whereas significantly inhibitory effects were observed when cells were incubated with Tat-NBD (WT). Consisting with p65 expression decrease analyzed by SABC method, NF-κB DNA-binding activity significantly decreased in Tat-NBD (WT) pretreatment group, especially at the largest dose. No significant changes were found in the control peptide group. CONCLUSION: Our result supports that active NF-κB participates in the pathogenesis of STC-induced acute pancreatitis in rats. Tat-NBD (WT) peptide has antiinflammatory effects in this model and inhibits the inflammation of acinus simulated by LPS.

  12. Truncated glucagon-like peptide-1 (proglucagon 78-107 amide), an intestinal insulin-releasing peptide, has specific receptors on rat insulinoma cells (RIN 5AH)

    DEFF Research Database (Denmark)

    Orskov, C; Nielsen, Jens Høiriis

    1988-01-01

    We studied binding of 125I-labelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide) to a cloned rat insulin-producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half-maximal binding was obtai......We studied binding of 125I-labelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide) to a cloned rat insulin-producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half-maximal binding...

  13. Targeting of liposomes to HIV-1-infected cells by peptides derived from the CD4 receptor.

    Science.gov (United States)

    Slepushkin, V A; Salem, I I; Andreev, S M; Dazin, P; Düzgüneş, N

    1996-10-23

    Liposomes can be targeted to HIV-infected cells by either reconstituting transmembrane CD4 in the membrane or covalently coupling soluble CD4 to modified lipids. We investigated whether synthetic peptides could be used as ligands for targeting liposomes. A synthetic peptide from the complementarity determining region 2 (CDR-2)-like domain of CD4 could bind specifically to HIV-infected cells and mediate the binding of peptide-coupled liposomes to these cells. A peptide from the CDR-3-like domain of CD4 inhibited HIV-induced syncytia formation, but failed to target liposomes to infected cells. This apparent discrepancy may be due to the requirement for a conformational change in the CD4 receptor for the CDR-3 region to interact with the HIV envelope protein. Our results demonstrate the feasibility of using synthetic peptides to target liposomes containing antiviral drugs to HIV-infected cells.

  14. Biofunctionalization of polycaprolactone scaffolds with RGD peptides for the better cells integration

    Science.gov (United States)

    Matveeva, V. G.; Seifalian, A. M.; Antonova, L. V.; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S.

    2016-08-01

    Here we tested in vitro electrospun polycaprolactone (PCL) scaffolds carbodiimide linkage with RGD peptides and their unconjugated counterparts. The scaffolds possessed highly porous structure and were formed by randomly distributed fibers. Orange II staining and ninhydrin test confirmed successful amination of the PCL. For the assessment of cell adhesion, we colonized scaffolds with primary human fibroblasts and counted the number of alive and dead cells. After 6 days of incubation, the number of fibroblasts on the scaffolds modified by RGD peptides significantly exceeded the number on unmodified scaffolds; however, the distribution of the cells on functionalized scaffolds was uneven, possibly due to uneven distribution of RGD peptides. The percentage of dead cells on the scaffolds with RGD peptides was significantly lower compared to their unmodified counterparts. Therefore, conjugation of PCL scaffolds with RGD peptides improves their integration with cells. This can be used in regenerative medicine.

  15. Interactions of Bio-Inspired Membranes with Peptides and Peptide-Mimetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Michael Sebastiano

    2015-08-01

    Full Text Available Via Dissipative Particle Dynamics (DPD and implicit solvent coarse-grained (CG Molecular Dynamics (MD we examine the interaction of an amphiphilic cell-penetrating peptide PMLKE and its synthetic counterpart with a bio-inspired membrane. We use the DPD technique to investigate the interaction of peptide-mimetic nanoparticles, or nanopins, with a three-component membrane. The CG MD approach is used to investigate the interaction of a cell-penetrating peptide PMLKE with single-component membrane. We observe the spontaneous binding and subsequent insertion of peptide and nanopin in the membrane by using CG MD and DPD approaches, respectively. In addition, we find that the insertion of peptide and nanopins is mainly driven by the favorable enthalpic interactions between the hydrophobic components of the peptide, or nanopin, and the membrane. Our study provides insights into the mechanism underlying the interactions of amphiphilic peptide and peptide-mimetic nanoparticles with a membrane. The result of this study can be used to guide the functional integration of peptide and peptide-mimetic nanoparticles with a cell membrane.

  16. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  17. RGDechi-hCit: αvβ3 selective pro-apoptotic peptide as potential carrier for drug delivery into melanoma metastatic cells.

    Directory of Open Access Journals (Sweden)

    Domenica Capasso

    Full Text Available αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.

  18. Study on the Penetrability of PEP-1-P27mt for Cell Membranes in Vitro

    Institute of Scientific and Technical Information of China (English)

    YAN Shirong; ZHU Minglei; QIU Fangcheng; WANG Lilin; QU Shen

    2007-01-01

    Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

  19. Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides.

    Science.gov (United States)

    Malanovic, Nermina; Lohner, Karl

    2016-05-01

    A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  20. Investigation of human cell response to covalently attached RADA16-I peptide on silicon surfaces.

    Science.gov (United States)

    Shamsi, Fahimeh

    2016-09-01

    We described a modification of the ionic (RADARADARADARADA)(1) peptide or RADA16-I with 4-azidophenyl isothiocyanate via a specific and gentle reaction. The azidated peptide was covalently immobilized on an alkyne-terminated monolayer on Si(111) via the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. Detailed characterization using Impedance spectroscopy (IS), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy demonstrated high coverage of the RADA 16-I peptide on silicon surfaces. Scanning electron microscopy (SEM) and methyl tetrazole sulfate (MTS) assay were used to characterize the morphology and proliferation ability of human fibroblast cells on surfaces. Cell adhesion assay was performed to examine cell-substrate interactions. Significant differences in fibroblast cell morphology, adhesion, and viability were observed on the RADA16-I peptide modified surfaces compared to the control surfaces. These results may suggest a potential application of RADA16-I peptide modified surfaces in biomedical applications. PMID:27236098

  1. Quantification of pharmaceutical peptides using selenium as an elemental detection label

    DEFF Research Database (Denmark)

    Møller, Laura Hyrup; Gabel-Jensen, Charlotte; Franzyk, Henrik;

    2014-01-01

    The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating p...

  2. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    Science.gov (United States)

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  3. The inability to process a self-peptide allows autoreactive T cells to escape tolerance

    OpenAIRE

    1993-01-01

    It is now clear that antigen presenting cells (APCs) do not present all the possible peptides of self-proteins to the immune system. When then, is the fate of T cells specific for those self-peptides that escape processing? In this study, the COOH-terminal peptide (residues 81-104) of self cytochrome c (cyt c) elicited strong autoimmune T cells, as well as autoantibodies specific for this immunogen. These T cells did not respond to stimulation with the whole self cyt c molecule, demonstrating...

  4. Short Peptides Enhance Single Cell Adhesion and Viability onMicroarrays

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Asphahani,Fareid; Zhang, Miqin

    2007-01-19

    Single cell patterning holds important implications forbiology, biochemistry, biotechnology, medicine, and bioinformatics. Thechallenge for single cell patterning is to produce small islands hostingonly single cells and retaining their viability for a prolonged period oftime. This study demonstrated a surface engineering approach that uses acovalently bound short peptide as a mediator to pattern cells withimproved single cell adhesion and prolonged cellular viabilityon goldpatterned SiO2 substrates. The underlying hypothesis is that celladhesion is regulated bythe type, availability, and stability ofeffective cell adhesion peptides, and thus covalently bound shortpeptides would promote cell spreading and, thus, single cell adhesion andviability. The effectiveness of this approach and the underlyingmechanism for the increased probability of single cell adhesion andprolonged cell viability by short peptides were studied by comparingcellular behavior of human umbilical cord vein endothelial cells on threemodelsurfaces whose gold electrodes were immobilized with fibronectin,physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently boundLys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and bindingproperties were characterized by reflectance Fourier transform infraredspectroscopy. Both short peptides were superior to fibronectin inproducing adhesion of only single cells, whereas the covalently boundpeptide also reduced apoptosis and necrosisof adhered cells. Controllingcell spreading by peptide binding domains to regulate apoptosis andviability represents a fundamental mechanism in cell-materialsinteraction and provides an effective strategy in engineering arrays ofsingle cells.

  5. Biological activity of Tat (47-58) peptide on human pathogenic fungi

    International Nuclear Information System (INIS)

    Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase

  6. Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Engl

    2013-10-01

    Full Text Available Christopher G England,1 Thomas Priest,2 Guandong Zhang,2 Xinghua Sun,2 Dhruvinkumar N Patel,2 Lacey R McNally,3,4 Victor van Berkel,4,5 André M Gobin,2 Hermann B Frieboes1,2,41Department of Pharmacology and Toxicology, 2Department of Bioengineering, 3Department of Medicine, 4James Graham Brown Cancer Center, 5Department of Surgery, University of Louisville, KY, USAAbstract: Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol (PEG-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.Keywords: cancer nanotherapy, tumor hypoxia, nanovector transport

  7. Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

    Science.gov (United States)

    Tixador, R.

    1992-01-01

    The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

  8. Thymoproteasomes produce unique peptide motifs for positive selection of CD8(+) T cells.

    Science.gov (United States)

    Sasaki, Katsuhiro; Takada, Kensuke; Ohte, Yuki; Kondo, Hiroyuki; Sorimachi, Hiroyuki; Tanaka, Keiji; Takahama, Yousuke; Murata, Shigeo

    2015-01-01

    Positive selection in the thymus provides low-affinity T-cell receptor (TCR) engagement to support the development of potentially useful self-major histocompatibility complex class I (MHC-I)-restricted T cells. Optimal positive selection of CD8(+) T cells requires cortical thymic epithelial cells that express β5t-containing thymoproteasomes (tCPs). However, how tCPs govern positive selection is unclear. Here we show that the tCPs produce unique cleavage motifs in digested peptides and in MHC-I-associated peptides. Interestingly, MHC-I-associated peptides carrying these tCP-dependent motifs are enriched with low-affinity TCR ligands that efficiently induce the positive selection of functionally competent CD8(+) T cells in antigen-specific TCR-transgenic models. These results suggest that tCPs contribute to the positive selection of CD8(+) T cells by preferentially producing low-affinity TCR ligand peptides.

  9. Effective Design of Multifunctional Peptides by Combining Compatible Functions.

    Science.gov (United States)

    Diener, Christian; Garza Ramos Martínez, Georgina; Moreno Blas, Daniel; Castillo González, David A; Corzo, Gerardo; Castro-Obregon, Susana; Del Rio, Gabriel

    2016-04-01

    Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.

  10. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

    OpenAIRE

    Battey, J F; Way, J M; Corjay, M H; Shapira, H; Kusano, K; Harkins, R.; Wu, J M; Slattery, T; Mann, E.; Feldman, R I

    1991-01-01

    The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor i...

  11. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

    Science.gov (United States)

    Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands. PMID:27626637

  12. Sequence-selective DNA binding with cell-permeable oligoguanidinium-peptide conjugates.

    Science.gov (United States)

    Mosquera, Jesús; Sánchez, Mateo I; Valero, Julián; de Mendoza, Javier; Vázquez, M Eugenio; Mascareñas, José L

    2015-03-21

    Conjugation of a short peptide fragment from a bZIP protein to an oligoguanidinium tail results in a DNA-binding miniprotein that selectively interacts with composite sequences containing the peptide-binding site next to an A/T-rich tract. In addition to stabilizing the complex with the target DNA, the oligoguanidinium unit also endows the conjugate with cell internalization properties.

  13. Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery

    Science.gov (United States)

    Gaspar, V. M.; Marques, J. G.; Sousa, F.; Louro, R. O.; Queiroz, J. A.; Correia, I. J.

    2013-07-01

    Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan-histidine-arginine (CH-H-R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

  14. Keratinocyte growth factor phage model peptides can promote epidermal cell proliferation without tumorigenic effect

    Institute of Scientific and Technical Information of China (English)

    ZONG Xian-lei; JIANG Du-yin; WANG Ji-chang; LIU Jun-li; LIU Zhen-zhong; CAI Jing-long

    2010-01-01

    Background Keratinocyte growth factor (KGF) significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight (56.9%) of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor (EGF). MTT assay data showed that four (No. 1-4) of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor (KGFR) mRNA in the KGF control group and the two phage model peptide groups (No. 1 and No. 2) increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups (No.1-4). Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.

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

    Directory of Open Access Journals (Sweden)

    Hong Yin

    2014-01-01

    Full Text Available 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.

  16. Design of embedded-hybrid antimicrobial peptides with enhanced cell selectivity and anti-biofilm activity.

    Directory of Open Access Journals (Sweden)

    Wei Xu

    Full Text Available Antimicrobial peptides have attracted considerable attention because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance which is the most common source of failure in bacterial infection treatment along with biofilms. The method to design hybrid peptide integrating different functional domains of peptides has many advantages. In this study, we designed an embedded-hybrid peptide R-FV-I16 by replacing a functional defective sequence RR7 with the anti-biofilm sequence FV7 embedded in the middle position of peptide RI16. The results demonstrated that the synthetic hybrid the peptide R-FV-I16 had potent antimicrobial activity over a wide range of Gram-negative and Gram-positive bacteria, as well as anti-biofilm activity. More importantly, R-FV-I16 showed lower hemolytic activity and cytotoxicity. Fluorescent assays demonstrated that R-FV-I16 depolarized the outer and the inner bacterial membranes, while scanning electron microscopy and transmission electron microscopy further indicated that this peptide killed bacterial cells by disrupting the cell membrane, thereby damaging membrane integrity. Results from SEM also provided evidence that R-FV-I16 inherited anti-biofilm activity from the functional peptide sequence FV7. Embedded-hybrid peptides could provide a new pattern for combining different functional domains and showing an effective avenue to screen for novel antimicrobial agents.

  17. In vitro cytocidal effect of lytic peptides on several transformed mammalian cell lines.

    Science.gov (United States)

    Jaynes, J M; Julian, G R; Jeffers, G W; White, K L; Enright, F M

    1989-01-01

    Several types of transformed mammalian cells, derived from established cell lines, were found to be lysed in vitro by three novel lytic peptides (SB-37, SB-37*, and Shiva-1). This is in contrast with the behavior of normal cells, where the observed lytic activity of the peptides is greatly reduced. Based on experiments utilizing compounds which disrupt the cytoskeleton (colchicine and cytochalasin-D), it is surmised that alterations in the cytoskeleton of transformed cells increase their sensitivity to the cytolytic activity exerted by the peptides, primarily by causing a loss of osmotic integrity. Thus, a stable and regenerative cytoskeletal system, as that possessed by normal cells, would seem requisite to withstanding the lytic effects of the peptides.

  18. Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators

    Directory of Open Access Journals (Sweden)

    David ePerez-Pascual

    2016-05-01

    Full Text Available Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence.

  19. Cytotoxic activity to acute myeloid leukemia cells by Antp-TPR hybrid peptide targeting Hsp90.

    Science.gov (United States)

    Horibe, Tomohisa; Kawamoto, Megumi; Kohno, Masayuki; Kawakami, Koji

    2012-07-01

    We previously reported that Antp-TPR hybrid peptide inhibited the interaction of Hsp90 with TPR2A and had selective cytotoxic activity discriminating between normal and cancer cells to induce cancer cell death. In this study, we investigated the cytotoxic activity of Antp-TPR peptide toward acute myeloid leukemia (AML) cells. It was demonstrated that Antp-TPR peptide induced AML cell death in cell lines such as U937, K562, THP-1, and HL-60 via activation of caspases 3 and 7, and disruption of mitochondrial membrane potential. Conversely, Antp-TPR peptide did not reduce the viability of normal cells including peripheral blood mononuclear cells (PBMCs), although both geldanamycin and 17-AAG, small-molecule inhibitors of Hsp90, mediated cytotoxicity to these normal cells at low concentrations. In addition, mutation analysis of TPR peptide demonstrated that the highly conserved amino acids Lys and Arg were critical to the cytotoxic activity. These results indicated that Antp-TPR hybrid peptide would provide potent and selective therapeutic options in the treatment of AML.

  20. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O;

    1996-01-01

    Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC c...

  1. A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells

    Directory of Open Access Journals (Sweden)

    Kawamoto Megumi

    2011-08-01

    Full Text Available Abstract Background Transferrin receptor (TfR is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. Methods In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. Results The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC50 values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC50 values > 50 μM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47

  2. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Science.gov (United States)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  3. Conservation analysis of dengue virust-cell epitope-based vaccine candidates using peptide block entropy

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Zhang, Guang Lan; Keskin, Derin B.;

    2011-01-01

    Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches are based on combinations of highly conserved T-cell epitopes. Peptide block entropy analysis is a novel approach for assembling sets....... In contrast, the benchmark study by Khan et al. (2008) resulted in 165 conserved 9-mer peptides. Many of the conserved blocks are located consecutively in the proteins. Connecting these blocks resulted in 78 conserved regions. Of the 1551 blocks of 9-mer peptides 110 comprised predicted HLA binder sets...

  4. Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells

    OpenAIRE

    Hutchinson, Tarun E.; Zhang, Jianliang; Xia, Shen-Ling; Kuchibhotla, Sudeep; Block, Edward R.; Patel, Jawaharlal M.

    2011-01-01

    We previously reported that the vasoactive peptide 1 (P1, “SSWRRKRKESS”) modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were...

  5. Detection of cancer cells using a peptide nanotube–folic acid modified graphene electrode

    DEFF Research Database (Denmark)

    Castillo, John J.; Svendsen, Winnie Edith; Rozlosnik, Noemi;

    2013-01-01

    This article describes the preparation of a graphene electrode modified with a new conjugate of peptide nanotubes and folic acid for the selective detection of human cervical cancer cells over-expressing folate receptors. The functionalization of peptide nanotubes with folic acid was confirmed by....... The modified electrode described here opens up new possibilities for future applications in early stage diagnoses of diseases where cells over-express folate receptors, such as in cancer or leishmaniasis disease....

  6. Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays.

    Directory of Open Access Journals (Sweden)

    Rok Seon Choung

    Full Text Available Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD by using a silicon-based peptide array and computational methods.Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes.Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity.These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.

  7. Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment, penetration, growth curve and direct cell-to-cell spread

    Institute of Scientific and Technical Information of China (English)

    SUN Zhaogang; ZHANG Manfu

    2005-01-01

    The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.

  8. The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

    Directory of Open Access Journals (Sweden)

    Romain Rivalin

    Full Text Available Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

  9. Follicular penetration and targeting.

    Science.gov (United States)

    Lademann, Jürgen; Otberg, Nina; Jacobi, Ute; Hoffman, Robert M; Blume-Peytavi, Ulrike

    2005-12-01

    In the past, intercellular penetration was assumed to be the most important penetration pathway of topically applied substances. First hints that follicular penetration needs to be taken into consideration were confirmed by recent investigations, presented during the workshop "Follicular Penetration and Targeting" at the 4th Intercontinental Meeting of Hair Research Societies", in Berlin 2004. Hair follicles represent an efficient reservoir for the penetration of topically applied substances with subsequent targeting of distinct cell populations, e.g., nestin-expressing follicular bulge cells. The volume of this reservoir can be determined by differential stripping technology. The follicular penetration processes are significantly influenced by the state of the follicular infundibulum; recent experimental investigations could demonstrate that it is essential to distinguish between open and closed hair follicles. Topically applied substances can only penetrate into open hair follicle. Knowledge of follicular penetration is of high clinical relevance for functional targeting of distinct follicular regions. Human hair follicles show a hair-cycle-dependent variation of the dense neuronal and vascular network. Moreover, during hair follicle cycling with initiation of anagen, newly formed vessels occur. Thus, the potential of nestin-expressing hair follicle stem cells to form neurons and blood vessels was investigated.

  10. Rearrangement of S-100 immunoreactive Langerhans' cells in human psoriatic skin treated with peptide T.

    Science.gov (United States)

    Wang, L; Hilliges, M; Talme, T; Marcusson, J A; Wetterberg, L; Johansson, O

    1995-01-01

    Dendritic cells marked by protein S-100 (S-100) antiserum in the suprabasal layers of the epidermis have previously been identified to be Langerhans' cells. In this study, S-100 immunoreactive cells have been investigated in psoriatic lesioned skin during and after peptide T treatment. Peptide T is an octapeptide with affinity for the CD4 receptor. Nine patients were intravenously infused with peptide T, 2 mg in 500 ml saline per day for 28 days. Sections from involved skin before, every week during, and after the treatment were processed by indirect immunofluorescence using S-100 antiserum. Before the treatment the epidermal Langerhans' cells were numerically decreased or even completely gone in the involved skin of psoriasis as compared to skin from normal healthy controls, while the dermal dendritic cells instead were increased and gathered in cell clusters around vascular structures. Four of the nine patients had histopathological improvements after the peptide T treatment, and, in those cases, the dendritic cells in the dermis were reduced in number, and the Langerhans' cells in the epidermis were numerically increased as well as even reversed to normal position and morphology. These changes in the distribution and density of Langerhans' cells represent their rearrangement during the course of psoriasis and/or the remission after peptide T treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7727353

  11. Turnover of Ia-peptide complexes is facilitated in viable antigen-presenting cells: biosynthetic turnover of Ia vs. peptide exchange.

    OpenAIRE

    Harding, C V; Roof, R W; Unanue, E R

    1989-01-01

    Macrophages and B cells process antigens to produce antigenic peptides that associate with class II major histocompatibility complex molecules (e.g., Ia molecules); these Ia-peptide complexes are recognized by CD4+ T lymphocytes. Processing of the antigen hen egg white lysozyme was inhibited by cycloheximide in peritoneal exudate cells (PECs, largely macrophages), but not in TA3 B-lymphoma cells. The uptake and metabolism of hen egg white lysozyme was largely intact in cycloheximide-treated P...

  12. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    Science.gov (United States)

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  13. Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides.

    Directory of Open Access Journals (Sweden)

    Jamie L Schafer

    2015-09-01

    Full Text Available Natural killer (NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs on NK cells and their major histocompatibility complex (MHC class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.

  14. Prospects for T cell immunotherapy of tumours by vaccination with immunodominant and subdominant peptides.

    Science.gov (United States)

    Melief, C J; Kast, W M

    1994-01-01

    Immunotherapy of tumours by adoptive transfer of cytotoxic T lymphocytes (CTL) is now feasible in experimental murine systems. These CTL recognize peptide sequences of defined length presented by major histocompatibility complex (MHC) class I molecules. Effective eradication of large tumour masses requires co-administration of interleukin 2. Tumour escape strategies are numerous but in various instances can be counteracted by defined measures. Initiation of CTL responses against poorly immunogenic virally induced tumours and other tumours requires novel strategies to overcome T cell inertia. We propose a strategy in which CTL are raised against target molecules of choice including differentiation antigens of restricted tissue distribution (autoantigens) or mutated/overexpressed oncogene products. The steps proposed include: (1) identification of target molecules of choice. (2) Identification in these target molecules of peptides fitting MHC allele-specific peptide motifs involved in peptide binding to MHC molecules. (3) Evaluation of actual binding of such peptides to specific MHC class I molecules. (4) In vitro CTL response induction by such peptides, presented by highly efficient antigen-presenting cells such as antigen processing-defective cells carrying empty MHC class I molecules loaded with a single peptide or dendritic cells. Both types of cells are capable of primary CTL response induction in vitro. (5) Evaluation of proper processing by the demonstration of tumour cell lysis by these CTL. (6) Adoptive transfer of tumour-specific CTL generated in vitro or vaccination with peptides. These various steps have now been taken for several viruses, virally induced tumours and other types of tumours and the first indications that this strategy is useful have been obtained. PMID:7796678

  15. Mechanism of cellular uptake of HIV-TAT peptide & effects of TAT-SOD against ultraviolet induced skin damage

    OpenAIRE

    Chen, Xiaochao

    2013-01-01

    TAT peptide is one of the best-characterised cell penetrating peptides (CPPs) derived from the transactivator of transcription protein from the human immunodeficiency virus 1 (HIV-1). TAT peptide is able to cross the cell membrane and deliver various biomolecules into cells with low immunogenicity and no toxicity. However, the exact mechanism of internalization still remains a subject of controversy. Lamellar neutron scattering was used to determine the location of TAT pepti...

  16. Antifungal Activity of 14-Helical β-Peptides against Planktonic Cells and Biofilms of Candida Species

    Directory of Open Access Journals (Sweden)

    Namrata Raman

    2015-08-01

    Full Text Available Candida albicans is the most prevalent cause of fungal infections and treatment is further complicated by the formation of drug resistant biofilms, often on the surfaces of implanted medical devices. In recent years, the incidence of fungal infections by other pathogenic Candida species such as C. glabrata, C. parapsilosis and C. tropicalis has increased. Amphiphilic, helical β-peptide structural mimetics of natural antimicrobial α-peptides have been shown to exhibit specific planktonic antifungal and anti-biofilm formation activity against C. albicans in vitro. Here, we demonstrate that β-peptides are also active against clinically isolated and drug resistant strains of C. albicans and against other opportunistic Candida spp. Different Candida species were susceptible to β-peptides to varying degrees, with C. tropicalis being the most and C. glabrata being the least susceptible. β-peptide hydrophobicity directly correlated with antifungal activity against all the Candida clinical strains and species tested. While β-peptides were largely ineffective at disrupting existing Candida biofilms, hydrophobic β-peptides were able to prevent the formation of C. albicans, C. glabrata, C. parapsilosis and C. tropicalis biofilms. The broad-spectrum antifungal activity of β-peptides against planktonic cells and in preventing biofilm formation suggests the promise of this class of molecules as therapeutics.

  17. Antifungal Activity of 14-Helical β-Peptides against Planktonic Cells and Biofilms of Candida Species.

    Science.gov (United States)

    Raman, Namrata; Lee, Myung-Ryul; Lynn, David M; Palecek, Sean P

    2015-01-01

    Candida albicans is the most prevalent cause of fungal infections and treatment is further complicated by the formation of drug resistant biofilms, often on the surfaces of implanted medical devices. In recent years, the incidence of fungal infections by other pathogenic Candida species such as C. glabrata, C. parapsilosis and C. tropicalis has increased. Amphiphilic, helical β-peptide structural mimetics of natural antimicrobial α-peptides have been shown to exhibit specific planktonic antifungal and anti-biofilm formation activity against C. albicans in vitro. Here, we demonstrate that β-peptides are also active against clinically isolated and drug resistant strains of C. albicans and against other opportunistic Candida spp. Different Candida species were susceptible to β-peptides to varying degrees, with C. tropicalis being the most and C. glabrata being the least susceptible. β-peptide hydrophobicity directly correlated with antifungal activity against all the Candida clinical strains and species tested. While β-peptides were largely ineffective at disrupting existing Candida biofilms, hydrophobic β-peptides were able to prevent the formation of C. albicans, C. glabrata, C. parapsilosis and C. tropicalis biofilms. The broad-spectrum antifungal activity of β-peptides against planktonic cells and in preventing biofilm formation suggests the promise of this class of molecules as therapeutics. PMID:26287212

  18. Quantitative & qualitative analysis of endothelial cells of donor cornea before & after penetrating keratoplasty in different pathological conditions

    Directory of Open Access Journals (Sweden)

    Aruna K.R. Gupta

    2016-01-01

    Full Text Available Background & objectives: Endothelial cells of the donor cornea are known to be affected quantitatively and qualitatively in different pathological conditions after penetrating keratoplasty (PK and this has direct effect on the clarity of vision obtained after PK. This study was undertaken to analyze the qualitative and quantitative changes in donor endothelial cells before and after PK in different pathological conditions. Methods: A prospective investigational analysis of 100 consecutive donor corneas used for penetrating keratoplasty between June 2006 and June 2008, was conducted. The patients were evaluated on the first day, at the end of first week, first month, third and six months and one year. Results: A decrease was observed in endothelial cell count in all pathological conditions. After one year of follow up the loss was 33.1 per cent in corneal opacity, 45.9 per cent in acute infective keratitis (AIK, 58.5 per cent in regrafts, 28.5 per cent in pseudophakic bullous keratopathy (PBK, 37 per cent in descemetocele, 27 per cent in keratoconus and 35.5 per cent in aphakic bullous keratopathy (ABK cases. Interpretation & conclusions: The endothelial cell loss was highest in regraft cases which was significant (P<0.05, while the least endothelial cell loss was seen in keratoconus cases. The cell loss was associated with increase in coefficient of variation (CV, i.e. polymegathism and pleomorphism. Inspite of this polymegathism and pleomorphism, the clarity of the graft was maintained.

  19. Targeting Antiapoptotic Bcl-2 Family Members with Cell-Permeable BH3 Peptides Induces Apoptosis Signaling, Death in Head, Neck Squamous Cell Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Rongxiu Li

    2007-10-01

    Full Text Available Head, neck squamous cell carcinomas (HNSCCs are frequently characterized by chemotherapy, radiation resistance, by overexpression of Bcl-XL, an antiapoptotic member of the Bcl-2 protein family. In this report, we examined whether cell-permeable peptides derived from the BH3 domains of proapoptotic Bax, Bad, or Bak could be used to target Bcl-XL and/or Bcl-2 in HNSCC cells, induce apoptotic death in these cells. To render the peptides cell-permeable, Antennapedia (Ant or polyarginine (R8 peptide transduction domain was fused to the amino termini. Fluorescence microscopy of peptide-treated HNSCC cells revealed that the BH3 peptides colocalized with mitochondria, the site of Bcl-XL, Bcl-2 expression. By contrast, a mutant peptide (BaxE BH3 that cannot bind Bcl-XL or Bcl-2 was diffusely localized throughout the cytoplasm. Treatment of three HNSCC cell lines (1483, UM-22A, UM-22B with the wild-type BH3 peptides resulted in loss of viability, induction of apoptosis, as assessed by 3-(4,5-dimethythiazol-2yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTS assays, annexin V staining. In general, Ant-conjugated peptides were more potent than R8-conjugated peptides, Bad BH3 peptide was typically more potent than Bax BH3 or Bak BH3. Treatment of purified HNSCC mitochondria with BH3 peptides resulted in robust release of cytochrome c. Thus, the relative apoptosis resistance of HNSCC cells is not due to a deficit in this step of the intrinsic, mitochondrialmediated apoptosis pathway. We conclude that cellpermeable BH3 peptides can be used to target Bcl-XL and/or Bcl-2 in HNSCC, that targeting of these proteins may have therapeutic value in the treatment of this disease.

  20. Screening and identification of a specific peptide for targeting hypoxic hepatoma cells.

    Science.gov (United States)

    Liu, Yiming; Xia, Xiangwen; Wang, Yong; Li, Xin; Zhou, Guofeng; Liang, Huiming; Feng, Gansheng; Zheng, Chuansheng

    2016-08-01

    The biological behaviors of residual hepatoma cells after transarterial embolization therapy, which exist in a hypoxic or even anaerobic tumor microenvironment, differ from the tumor cells under normoxic conditions. This study aimed to use a phage display peptide library for in vivo and in vitro screening to obtain a peptide which could specifically bind to hypoxic hepatoma cells, allowing further targeted diagnosis and treatment for liver cancer. In this study, hypoxic hepatoma cells HepG2 (targeted cells), and normal liver cells HL-7702 (control cells), were utilized to perform three rounds of in vitro screening using a phage-displayed 7-mer peptide library. In addition, hypoxic HepG2 were subcutaneously injected into nude mice to establish a hepatocarcinoma model, followed by performing three rounds of in vivo screening on the phages identified from the in vitro screening. The products from the screening were further identified using ELISA and immunofluorescence staining on cells and tissues. The results indicated that the P11 positive clone had the highest binding effect with hypoxic hepatoma cells. The sequence of the exogenous insert fragment of P11 positive clone was obtained by sequencing: GSTSFSK. The binding assay indicated that GSTSFSK could specifically bind to hypoxic hepatoma cells and hepatocarcinoma tissues. This 7-mer peptide has the potential to be developed as an useful molecular to the targeting diagnosis and treatment of residual hepatoma cells after transarterial chemoembolization. PMID:27381416

  1. C-peptide protects against hyperglycemic memory and vascular endothelial cell apoptosis.

    Science.gov (United States)

    Bhatt, Mahendra Prasad; Lee, Yeon-Ju; Jung, Se-Hui; Kim, Yong Ho; Hwang, Jong Yun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Young-Myeong; Ha, Kwon-Soo

    2016-10-01

    C-peptide exerts protective effects against diabetic complications; however, its role in inhibiting hyperglycemic memory (HGM) has not been elucidated. We investigated the beneficial effect of C-peptide on HGM-induced vascular damage in vitro and in vivo using human umbilical vein endothelial cells and diabetic mice. HGM induced apoptosis by persistent generation of intracellular ROS and sustained formation of ONOO(-) and nitrotyrosine. These HGM-induced intracellular events were normalized by treatment with C-peptide, but not insulin, in endothelial cells. C-peptide also inhibited persistent upregulation of p53 and activation of mitochondrial adaptor p66(shc) after glucose normalization. Further, C-peptide replacement therapy prevented persistent generation of ROS and ONOO(-) in the aorta of diabetic mice whose glucose levels were normalized by the administration of insulin. C-peptide, but not insulin, also prevented HGM-induced endothelial apoptosis in the murine diabetic aorta. This study highlights a promising role for C-peptide in preventing HGM-induced intracellular events and diabetic vascular damage.

  2. Self-assembling tryptophan-based designer peptides as intracellular delivery vehicles.

    Science.gov (United States)

    Bhardwaj, Ishanki; Jha, Divya; Admane, Prasad; Panda, Amulya K; Haridas, V

    2016-01-15

    A series of tryptophan-based peptides W1a, b-W4a, b, with diverse architectures were designed and synthesized. These tryptophan containing peptides can self-assemble to spherical particle. This self-assembled system was demonstrated to encapsulate rhodamine B and penetrate the cell membrane.

  3. Immobilization of RGD Peptides onto Decellularized Valve Scaffolds to Promote Cell Adhesion

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD(arginine-glycine-aspartic acid) containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

  4. Natriuretic peptide receptor-3 underpins the disparate regulation of endothelial and vascular smooth muscle cell proliferation by C-type natriuretic peptide

    OpenAIRE

    Khambata, Rayomand S.; Panayiotou, Catherine M; Hobbs, Adrian J

    2011-01-01

    BACKGROUND AND PURPOSE C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the Gi-coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis. EXPERIMENT...

  5. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  6. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Science.gov (United States)

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  7. Photoinduced apoptosis using a peptide carrying a photosensitizer.

    Science.gov (United States)

    Watanabe, Kazunori; Fujiwara, Hayato; Kitamatsu, Mizuki; Ohtsuki, Takashi

    2016-07-01

    A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines. PMID:27165853

  8. A family of cell-adhering peptides homologous to fibrinogen C-termini

    International Nuclear Information System (INIS)

    Research highlights: → Cell-adhesive sequences homologous to fibrinogen C-termini exist in other proteins. → The extended homologous cell-adhesive C-termini peptides family is termed Haptides. → In membrane-like environment random coiled Haptides adopt a helical conformation. → Replacing positively charged residues with alanine reduces Haptides activity. -- Abstract: A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides Cβ, preCγ, and CαE, corresponding to sequences on the C-termini of fibrinogen chains β, γ, and αE, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1 and 2, tenascins C and X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preCγ peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

  9. Synchrotron X-ray fluorescence studies of a bromine-labelled cyclic RGD peptide interacting with individual tumor cells

    International Nuclear Information System (INIS)

    The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells. The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells

  10. Shaping T Cell – B Cell Collaboration in the Response to Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 by Peptide Priming

    Science.gov (United States)

    Steede, N. Kalaya; Rust, Blake J.; Hossain, Mohammad M.; Freytag, Lucy C.; Robinson, James E.; Landry, Samuel J.

    2013-01-01

    Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming. PMID:23776539

  11. Shaping T cell - B cell collaboration in the response to human immunodeficiency virus type 1 envelope glycoprotein gp120 by peptide priming.

    Directory of Open Access Journals (Sweden)

    N Kalaya Steede

    Full Text Available Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.

  12. Peptide-pulsed dendritic cells have superior ability to induce immune-mediated tissue destruction compared to peptide with adjuvant.

    Directory of Open Access Journals (Sweden)

    Dilan Dissanayake

    Full Text Available Vaccines for cancer immunotherapy are of interest but in general have not yet achieved the desired therapeutic efficacy in clinical trials. We present here a novel model to evaluate vaccine strategies by following tissue destruction in a transgenic model, where a defined antigen is expressed on pancreatic islets. We found that the transfer of syngeneic antigen-pulsed dendritic cells (DCs resulted in autoimmune cytotoxic T-lymphocyte activation that was not observed following vaccinations that were based on peptides and adjuvants. Importantly, the induction of diabetes by DC transfer is dependent upon the maturation of DCs prior to transfer. Furthermore, diabetes induction only occurred if DCs were pulsed with the immunodominant epitope in addition to at least one other peptide, suggesting greater cytolytic activity upon engagement of multiple T-cell specificities. While the tumor environment undoubtedly will be more complex than healthy tissue, the insights gained through this model provide useful information on variables that can affect CD8-mediated tissue cytolysis in vivo.

  13. Cell-collagen interactions : the use of peptide Toolkits to investigate collagen-receptor interactions

    NARCIS (Netherlands)

    Farndale, Richard W.; Lisman, Ton; Bihan, Dominique; Hamaia, Samir; Smerling, Christiane S.; Pugh, Nicholas; Konitsiotis, Antonios; Leitinger, Birgit; de Groot, Philip G.; Jarvis, Gavin E.; Raynal, Nicolas

    2008-01-01

    Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptide

  14. A smart tumor targeting peptide-drug conjugate, pHLIP-SS-DOX: synthesis and cellular uptake on MCF-7 and MCF-7/Adr cells.

    Science.gov (United States)

    Song, Qin; Chuan, Xingxing; Chen, Binlong; He, Bing; Zhang, Hua; Dai, Wenbing; Wang, Xueqing; Zhang, Qiang

    2016-06-01

    Doxorubicin (DOX) is a potent anticancer drug for the treatment of tumors, but the poor specificity and multi-drug resistance (MDR) on tumor cells have restricted its application. Here, a pH and reduction-responsive peptide-drug conjugate (PDC), pHLIP-SS-DOX, was synthesized to overcome these drawbacks. pH low insertion peptide (pHLIP) is a cell penetrating peptide (CPP) with pH-dependent transmembrane ability. And because of the unique cell membrane insertion pattern, it might reverse the MDR. The cellular uptake study showed that on both drug-sensitive MCF-7 and drug-resistant MCF-7/Adr cells, pHLIP-SS-DOX obviously facilitated the uptake of DOX at pH 6.0 and the uptake level on MCF-7/Adr cells was similar with that on MCF-7 cells, indicating that pHLIP-SS-DOX had the ability to target acidic tumor cells and reverse MDR. In vitro cytotoxicity study mediated by GSH-OEt demonstrated that the cytotoxic effect of pHLIP-SS-DOX was reduction responsive, with obvious cytotoxicity at pH 6.0; while it had poor cytotoxicity at pH 7.4, no matter with or without GSH-OEt pretreatment. This illustrated that pHLIP could deliver DOX into tumor cells with acidic microenvironment specifically and could not deliver drugs into normal cells with neutral microenvironment. In summary, pHLIP-SS-DOX is a promising strategy to target drugs to tumors and provides a possibility to overcome MDR.

  15. Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

    Directory of Open Access Journals (Sweden)

    Jamie L Brugnano

    Full Text Available In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2, enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy.

  16. Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

    Science.gov (United States)

    Brugnano, Jamie L; Panitch, Alyssa

    2014-01-01

    In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy.

  17. REDV Peptide Conjugated Nanoparticles/pZNF580 Complexes for Actively Targeting Human Vascular Endothelial Cells.

    Science.gov (United States)

    Shi, Changcan; Li, Qian; Zhang, Wencheng; Feng, Yakai; Ren, Xiangkui

    2015-09-16

    Herein, we demonstrate that the REDV peptide modified nanoparticles (NPs) can serve as a kind of active targeting gene carrier to condensate pZNF580 for specific promotion of the proliferation of endothelial cells (ECs). First, we synthesized a series of biodegradable amphiphilic copolymers by ring-opening polymerization reaction and graft modification with REDV peptide. Second, we prepared active targeting NPs via self-assembly of the amphiphilic copolymers using nanoprecipitation technology. After condensation with negatively charged pZNF580, the REDV peptide modified NPs/pZNF580 complexes were formed finally. Due to the binding affinity toward ECs of the specific peptide, these REDV peptide modified NPs/pZNF580 complexes could be recognized and adhered specifically by ECs in the coculture system of ECs and human artery smooth muscle cells (SMCs) in vitro. After expression of ZNF580, as the key protein to promote the proliferation of ECs, the relative ZNF580 protein level increased from 15.7% to 34.8%. The specificity in actively targeting ECs of the REDV peptide conjugated NPs/pZNF580 complexes was still retained in the coculture system. These findings in the present study could facilitate the development of actively targeting gene carriers for the endothelialization of artificial blood vessels. PMID:26373583

  18. Phosphorylated Peptide Functionalization of Lanthanide Upconversion Nanoparticles for Tuning Nanomaterial-Cell Interactions.

    Science.gov (United States)

    Yao, Chi; Wei, Caiyi; Huang, Zhi; Lu, Yiqing; El-Toni, Ahmed Mohamed; Ju, Dianwen; Zhang, Xiangmin; Wang, Wenning; Zhang, Fan

    2016-03-23

    Peptide modification of nanoparticles with high efficiency is critical in determining the properties and bioapplications of nanoparticles, but the methodology remains a challenging task. Here, by using the phosphorylated linear and cyclic peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the peptides binding efficiency for the inorganic metal compound nanoparticles was increased significantly after the phosphorylation treatment, and the modification allowed for improving the selectivity and signal-to-noise ratio for cancer targeting and reduced the toxicity derived from nonspecific interactions of nanoparticles with cells owing to the higher amount of phosphopeptide binding. In addition, molecular dynamics (MD) simulations of various peptides on inorganic metal compound surfaces revealed that the peptide adsorption on the surface is mainly driven by electrostatic interactions between phosphate oxygen and the polarized interfacial water layer, consistent with the experimental observation of the strong binding propensity of phosphorylated peptides. Significantly, with the RGD phosphopeptide surface modification, these nanoparticles provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy and may prove useful for various diagnostic and therapeutic applications.

  19. Phosphorylated Peptide Functionalization of Lanthanide Upconversion Nanoparticles for Tuning Nanomaterial-Cell Interactions.

    Science.gov (United States)

    Yao, Chi; Wei, Caiyi; Huang, Zhi; Lu, Yiqing; El-Toni, Ahmed Mohamed; Ju, Dianwen; Zhang, Xiangmin; Wang, Wenning; Zhang, Fan

    2016-03-23

    Peptide modification of nanoparticles with high efficiency is critical in determining the properties and bioapplications of nanoparticles, but the methodology remains a challenging task. Here, by using the phosphorylated linear and cyclic peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the peptides binding efficiency for the inorganic metal compound nanoparticles was increased significantly after the phosphorylation treatment, and the modification allowed for improving the selectivity and signal-to-noise ratio for cancer targeting and reduced the toxicity derived from nonspecific interactions of nanoparticles with cells owing to the higher amount of phosphopeptide binding. In addition, molecular dynamics (MD) simulations of various peptides on inorganic metal compound surfaces revealed that the peptide adsorption on the surface is mainly driven by electrostatic interactions between phosphate oxygen and the polarized interfacial water layer, consistent with the experimental observation of the strong binding propensity of phosphorylated peptides. Significantly, with the RGD phosphopeptide surface modification, these nanoparticles provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy and may prove useful for various diagnostic and therapeutic applications. PMID:26927957

  20. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling.

    Directory of Open Access Journals (Sweden)

    Zhongqiu Ni

    Full Text Available A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl phenyl-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.

  1. Gastrin releasing peptide GRP(14-27) in human breast cancer cells and in small cell lung cancer

    DEFF Research Database (Denmark)

    Vangsted, A J; Andersen, E V; Nedergaard, L;

    1991-01-01

    % of the samples. When the GRP(14-27) peptide was added exogenously to breast cancer and SCLC cell lines under serum-free culture conditions, (3H)-thymidine incorporation was stimulated by GRP(14-27) in the SCLC cell lines. Of the breast cancer cell lines only the T47D cell line responded with an increase in (3H......Immunoreactivity related to the gastrin-releasing peptide (GRP) precursor was detected in four different human breast cancer cell lines. The amounts and the characteristics in extracts from different breast carcinoma cells were compared with cell extracts from small cell lung cancer (SCLC) cells......(14-27) or GRP(18-27) in Sephadex G-50 chromatography. No immunoreactivity was detected in the fractions containing high molecular weight components. In a total of 41 human breast carcinoma biopsies from different postmenopausal patients, IR-GRP was detected by immunohistological staining in 39...

  2. Triple Effect of Mimetic Peptides Interfering with Neural Cell Adhesion Molecule Homophilic Cis Interactions

    DEFF Research Database (Denmark)

    Li, S. Z.; Kolkova, Kateryna; Rudenko, Olga;

    2005-01-01

    The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects...... on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate...... of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B...

  3. Core-shell nanoparticle-based peptide therapeutics and combined hyperthermia for enhanced cancer cell apoptosis.

    Science.gov (United States)

    Shah, Birju P; Pasquale, Nicholas; De, Gejing; Tan, Tao; Ma, Jianjie; Lee, Ki-Bum

    2014-09-23

    Mitochondria-targeting peptides have garnered immense interest as potential chemotherapeutics in recent years. However, there is a clear need to develop strategies to overcome the critical limitations of peptides, such as poor solubility and the lack of target specificity, which impede their clinical applications. To this end, we report magnetic core-shell nanoparticle (MCNP)-mediated delivery of a mitochondria-targeting pro-apoptotic amphipathic tail-anchoring peptide (ATAP) to malignant brain and metastatic breast cancer cells. Conjugation of ATAP to the MCNPs significantly enhanced the chemotherapeutic efficacy of ATAP, while the presence of targeting ligands afforded selective delivery to cancer cells. Induction of MCNP-mediated hyperthermia further potentiated the efficacy of ATAP. In summary, a combination of MCNP-mediated ATAP delivery and subsequent hyperthermia resulted in an enhanced effect on mitochondrial dysfunction, thus resulting in increased cancer cell apoptosis. PMID:25133971

  4. Antigen-oriented T cell migration contributes to myelin peptide induced-EAE and immune tolerance.

    Science.gov (United States)

    Zheng, Peiguo; Fu, Hanxiao; Wei, Gaohui; Wei, Zhongwei; Zhang, Junhua; Ma, Xuehan; Rui, Dong; Meng, Xianchun; Ming, Liang

    2016-08-01

    Treatment with soluble myelin peptide can efficiently and specifically induce tolerance to demyelination autoimmune diseases including multiple sclerosis, however the mechanism underlying this therapeutic effect remains to be elucidated. In actively induced mouse model of experimental autoimmune encephalomyelitis (EAE) we analyzed T cell and innate immune cell responses in the central nervous system (CNS) and spleen after intraperitoneal (i.p.) infusion of myelin oligodendrocyte glycoprotein (MOG). We found that i.p. MOG infusion blocked effector T cell recruitment to the CNS and protected mice from EAE and lymphoid organ atrophy. Innate immune CD11b(+) cells preferentially recruited MOG-specific effector T cells, particularly when activated to become competent antigen presenting cells (APCs). During EAE development, mature APCs were enriched in the CNS rather than in the spleen, attracting effector T cells to the CNS. Increased myelin antigen exposure induced CNS-APC maturation, recruiting additional effector T cells to the CNS, causing symptoms of disease. MOG triggered functional maturation of splenic APCs. MOG presenting APCs interacted with MOG-specific T cells in the spleen, aggregating to cluster around CD11b(+) cells, and were trapped in the periphery. This process was MHC II dependent as an MHC II directed antibody blocked CD4(+) T cell cluster formation. These findings highlight the role of myelin peptide-loaded APCs in myelin peptide-induced EAE and immune tolerance. PMID:27327113

  5. Cyclic Dipeptide Shuttles as a Novel Skin Penetration Enhancement Approach: Preliminary Evaluation with Diclofenac.

    Science.gov (United States)

    Mohammed, Yousuf; Teixidó, Meritxell; Namjoshi, Sarika; Giralt, Ernest; Benson, Heather

    2016-01-01

    This study demonstrates the effectiveness of a peptide shuttle in delivering diclofenac into and through human epidermis. Diclofenac was conjugated to a novel phenylalanyl-N-methyl-naphthalenylalanine-derived diketopiperazine (DKP) shuttle and to TAT (a classical cell penetrating peptide), and topically applied to human epidermis in vitro. DKP and TAT effectively permeated into and through human epidermis. When conjugated to diclofenac, both DKP and TAT enhanced delivery into and through human epidermis, though DKP was significantly more effective. Penetration of diclofenac through human epidermis (to receptor) was increased by conjugation to the peptide shuttle and cell penetrating peptide with enhancement of 6x by DKP-diclofenac and 3x by TAT-diclofenac. In addition, the amount of diclofenac retained within the epidermis was significantly increased by peptide conjugation. COX-2 inhibition activity of diclofenac was retained when conjugated to DKP. Our study suggests that the peptide shuttle approach may offer a new strategy for targeted delivery of small therapeutic and diagnostic molecules to the skin. PMID:27548780

  6. Cyclic Dipeptide Shuttles as a Novel Skin Penetration Enhancement Approach: Preliminary Evaluation with Diclofenac

    Science.gov (United States)

    Namjoshi, Sarika; Giralt, Ernest; Benson, Heather

    2016-01-01

    This study demonstrates the effectiveness of a peptide shuttle in delivering diclofenac into and through human epidermis. Diclofenac was conjugated to a novel phenylalanyl-N-methyl-naphthalenylalanine-derived diketopiperazine (DKP) shuttle and to TAT (a classical cell penetrating peptide), and topically applied to human epidermis in vitro. DKP and TAT effectively permeated into and through human epidermis. When conjugated to diclofenac, both DKP and TAT enhanced delivery into and through human epidermis, though DKP was significantly more effective. Penetration of diclofenac through human epidermis (to receptor) was increased by conjugation to the peptide shuttle and cell penetrating peptide with enhancement of 6x by DKP-diclofenac and 3x by TAT-diclofenac. In addition, the amount of diclofenac retained within the epidermis was significantly increased by peptide conjugation. COX-2 inhibition activity of diclofenac was retained when conjugated to DKP. Our study suggests that the peptide shuttle approach may offer a new strategy for targeted delivery of small therapeutic and diagnostic molecules to the skin. PMID:27548780

  7. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

    OpenAIRE

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven CL; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC ...

  8. HIV-1 Capsid Assembly Inhibitor (CAI) Peptide: Structural Preferences and Delivery into Human Embryonic Lung Cells and Lymphocytes

    OpenAIRE

    Braun, Klaus; Frank, Martin; Pipkorn, Rüdiger; Reed, Jennifer; Spring, Herbert; Debus, Jürgen; Didinger, Bernd; von der Lieth, Claus-Wilhelm; Wiessler, Manfred; Waldeck, Waldemar

    2008-01-01

    The Human immunodeficiency virus 1 derived capsid assembly inhibitor peptide (HIV-1 CAI-peptide) is a promising lead candidate for anti-HIV drug development. Its drawback, however, is that it cannot permeate cells directly. Here we report the transport of the pharmacologically active CAI-peptide into human lymphocytes and Human Embryonic Lung cells (HEL) using the BioShuttle platform. Generally, the transfer of pharmacologically active substances across membranes, demonstrated by confocal las...

  9. HIV-1 Capsid Assembly Inhibitor (CAI) Peptide: Structural Preferences and Delivery into Human Embryonic Lung Cells and Lymphocytes

    OpenAIRE

    Klaus Braun, Martin Frank, Rüdiger Pipkorn, Jennifer Reed, Herbert Spring, Jürgen Debus, Bernd Didinger, Claus-Wilhelm von der Lieth, Manfred Wiessler, Waldemar Waldeck

    2008-01-01

    The Human immunodeficiency 1 derived capsid assembly inhibitor peptide (HIV-1 CAI-peptide) is a promising lead candidate for anti-HIV drug development. Its drawback, however, is that it cannot permeate cells directly. Here we report the transport of the pharmacologically active CAI-peptide into human lymphocytes and Human Embryonic Lung cells (HEL) using the BioShuttle platform. Generally, the transfer of pharmacologically active substances across membranes, demonstrated by confocal laser sca...

  10. The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

    Directory of Open Access Journals (Sweden)

    Dimitris Missirlis

    Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

  11. Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells.

    Science.gov (United States)

    Zhou, Fang; Jia, Xiaoling; Yang, Yang; Yang, Qingmao; Gao, Chao; Zhao, Yunhui; Fan, Yubo; Yuan, Xiaoyan

    2016-11-01

    The efficiency of biomaterials used in small vascular repair depends greatly on their ability to interact with vascular endothelial cells (VECs). Rapid endothelialization of the vascular grafts is a promising way to prevent thrombosis and intimal hyperplasia. In this work, modification of electrospun membranes of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) by three different peptides for regulation of VECs were studied in order to obtain ideal bioactive biomaterials as small diameter vascular grafts. QK (a mimetic peptide to vascular endothelial growth factor), Arg-Glu-Asp-Val (REDV, a specific adhesive peptide to VECs) and Val-Ala-Pro-Gly (VAPG, a specific adhesive peptide to vascular smooth muscle cells) were investigated. Surface properties of the modified membranes and the response of VECs were verified. It was found that protein adsorption and platelet adhesion were effectively suppressed with the introduction of QK, REDV or VAPG peptides on the PELCL electrospun membranes. Both QK- and REDV-modified electrospun membranes could accelerate the proliferation of VECs in the first 9days, and the QK-modified electrospun membrane promoted cell proliferation more significantly than the REDV-modified one. The REDV-modified PELCL membrane was the most favorable for VECs adhesion than QK- and VAPG-modified membranes. It was suggested that QK- or REDV-modified PELCL electrospun membranes may have great potential applications in cardiovascular biomaterials for rapid endothelialization in situ. PMID:27524062

  12. TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors.

    Science.gov (United States)

    Doorduijn, Elien M; Sluijter, Marjolein; Querido, Bianca J; Oliveira, Cláudia C; Achour, Adnane; Ossendorp, Ferry; van der Burg, Sjoerd H; van Hall, Thorbald

    2016-02-01

    Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the "self" TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies. PMID:26784543

  13. TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors

    Science.gov (United States)

    Doorduijn, Elien M.; Sluijter, Marjolein; Querido, Bianca J.; Oliveira, Cláudia C.; Achour, Adnane; Ossendorp, Ferry; van der Burg, Sjoerd H.; van Hall, Thorbald

    2016-01-01

    Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies. PMID:26784543

  14. Molecular characterization of antigen-peptide pulsed dendritic cells: immature dendritic cells develop a distinct molecular profile when pulsed with antigen peptide.

    Directory of Open Access Journals (Sweden)

    Amy X Yang

    Full Text Available As dendritic cells (DCs are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC, immature DC (iDC, and mature DC (mDC from human peripheral blood monocytes (PBMC obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20, a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC. When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA, pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN, anti-apoptosis (NFKBIA, DNA repair (MRPS11, RAD21, TXNRD1, and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61 in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host.

  15. Enhancement of cytotoxicity of antimicrobial peptide magainin Ⅱ in tumor cells by bombesin-targeted delivery

    Institute of Scientific and Technical Information of China (English)

    Shan LIU; Hao YANG; Lin WAN; Hua-wei CAI; Sheng-fu LI; You-ping LI; Jing-qiu CHENG; Xiao-feng LU

    2011-01-01

    Aim: To investigate whether the conjugation of magainin II(MG2),an antimicrobial peptides(AMPs),to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells.Methods: A magainin Ⅱ-bombesin conjugate(MG2B)was constructed by attaching magainin Ⅱ(MG2)to bombesin at its N-terminus.The peptides were synthesized using Fmoc-chemistry.The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 celt counting kit.Moreover,the in vivo antitumor effect of the peptide was determined in tumor xenograft models.Results: The IC50 of MG2B for cancer cells(10-15 μmol/L)was at least 10 times lower than the IC50 of unconjugated MG2(125μmol/L).Moreover,the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2.In contrast,conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2,suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding.Indeed,MG2B selectively induced cell death in cancer cells in vitro with the IC50 ranging from 10 to 15 μmol/L,which was about 6-10 times lower than the IC50 for normal cells.MG2B(20mg/kg per day,intratumorally injected for 5 d)also exhibited antitumor effects in mice bearing MCF-7 tumor grafts.The mean weights of tumor grafts in MG2B-and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g,respectively.Conclusion: The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.

  16. In silico design of discontinuous peptides representative of B and T-cell epitopes from HER2-ECD as potential novel cancer peptide vaccines.

    Science.gov (United States)

    Manijeh, Mahdavi; Mehrnaz, Keyhanfar; Violaine, Moreau; Hassan, Mohabatkar; Abbas, Jafarian; Mohammad, Rabbani

    2013-01-01

    At present, the most common cause of cancer-related death in women is breast cancer. In a large proportion of breast cancers, there is the overexpression of human epidermal growth factor receptor 2 (HER2). This receptor is a 185 KDa growth factor glycoprotein, also known as the first tumor-associated antigen for different types of breast cancers. Moreover, HER2 is an appropriate cell-surface specific antigen for passive immunotherapy, which relies on the repeated application of monoclonal antibodies that are transferred to the patient. However, vaccination is preferable because it would stimulate a patient's own immune system to actively respond to a disease. In the current study, several bioinformatics tools were used for designing synthetic peptide vaccines. PEPOP was used to predict peptides from HER2 ECD subdomain III in the form of discontinuous-continuous B-cell epitopes. Then, T-cell epitope prediction web servers MHCPred, SYFPEITHI, HLA peptide motif search, Propred, and SVMHC were used to identify class-I and II MHC peptides. In this way, PEPOP selected 12 discontinuous peptides from the 3D structure of the HER2 ECD subdomain III. Furthermore, T-cell epitope prediction analyses identified four peptides containing the segments 77 (384-391) and 99 (495-503) for both B and T-cell epitopes. This work is the only study to our knowledge focusing on design of in silico potential novel cancer peptide vaccines of the HER2 ECD subdomain III that contain epitopes for both B and T-cells. These findings based on bioinformatics analyses may be used in vaccine design and cancer therapy; saving time and minimizing the number of tests needed to select the best possible epitopes.

  17. Cell- and nuclear-penetrating anti-dsDNA autoantibodies have multiple arginines in CDR3 of VH and increase cellular level of pERK and Bcl-2 in mesangial cells.

    Science.gov (United States)

    Im, Sae-Ran; Im, Sun-Woo; Chung, Hee-Yong; Pravinsagar, Pavithra; Jang, Young-Ju

    2015-10-01

    Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules.

  18. Milk peptides increase iron solubility in water but do not affect DMT-1 expression in Caco-2 cells

    Science.gov (United States)

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. Our objectives were to investigate whether these fractions a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells b) enhance iron dialyzability when added in meals. Peptid...

  19. T-Cell Recognition of Mycobacterial GroES Peptides in Thai Leprosy Patients and Contacts

    Science.gov (United States)

    Chua-Intra, Boosbun; Peerapakorn, Somchai; Davey, Nick; Jurcevic, Stipo; Busson, Marc; Vordermeier, H. Martin; Pirayavaraporn, Charoon; Ivanyi, Juraj

    1998-01-01

    We report here the mapping of T-cell-stimulatory determinants of the GroES 10-kDa heat shock protein homologues from Mycobacterium leprae and Mycobacterium tuberculosis, which are known as major immunogens in mycobacterial infections. Peripheral blood mononuclear cells (PBMC) from treated tuberculoid leprosy or lepromatous leprosy patients and from healthy household or hospital staff contacts of the patients were cultured with 20 16-mer peptides covering the entire sequences of both M. leprae and M. tuberculosis GroES. The total number of recognized peptides was found to be the largest in family contacts, while responder frequencies to the individual tested peptides varied (5 to 80%) with specificity between the patient and contact groups. Proliferative responses to some peptides showed positive or negative associations of low statistical significance with DR and DQ alleles, though responses to most GroES peptides were genetically permissive. Notably, the sequence of the 25–40 peptide of M. leprae, but not that of M. tuberculosis, was more frequently stimulatory in tuberculoid leprosy patients than in either group of sensitized healthy contacts. This peptide bound to a number of HLA-DR molecules, of which HLA-DRB5*0101 had the strongest affinity. The epitope core binding to this allele was localized to the 29-to-37 sequence, and its key residue was localized to the M. leprae-specific glutamic acid at position 32. This epitope may be of interest for the development of a blood test- or skin test-based diagnostic reagent for tuberculoid leprosy, subject to further clinical evaluation in untreated patients. PMID:9746595

  20. HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4(+) T Cell Responses

    DEFF Research Database (Denmark)

    Wang, M. J.; Larsen, Mette Voldby; Nielsen, Morten;

    2010-01-01

    Background: Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. Methodology/Principal Findings: In the present work, bioinformatics was used to predict...... synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFN gamma ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder...... cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence...

  1. Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

    Institute of Scientific and Technical Information of China (English)

    Yi Peng WANG; Ze Fen WANG; Ying Chun ZHANG; Qing TIAN; Jian Zhi WANG

    2004-01-01

    Abnormal deposition of amyloid-β(Aβ) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer's disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/Aβ and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of Aβ on the inhibition of cell differentiation,we added exogenously the similar level or about 10-times of the Aβ level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawalinduced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of Aβ into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ1-42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of Aβ could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.

  2. Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response.

    Directory of Open Access Journals (Sweden)

    Antonella Capozzi

    Full Text Available Celiac Disease (CD is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF, which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 α-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2 activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10-mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease.

  3. Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice

    NARCIS (Netherlands)

    Ellenbroek, J.H.; Tons, H.A.; Westerouen van Meeteren, M.J.; de Graaf, N.; Hanegraaf, M.A.; Rabelink, T.J.; Carlotti, F.; de Koning, E.J.

    2013-01-01

    AIMS/HYPOTHESIS: Incretin-based therapies improve glycaemic control in patients with type 2 diabetes. In animal models of diabetes, glucagon-like peptide-1 receptor agonists (GLP-1RAs) increase beta cell mass. GLP-1RAs are also evaluated in non-diabetic individuals with obesity and cardiovascular di

  4. Cinacalcet for hypercalcemia caused by pulmonary squamous cell carcinoma producing parathyroid hormone-related Peptide

    NARCIS (Netherlands)

    Bech, A.; Smolders, K.; Telting, D.; Boer, H. de

    2012-01-01

    BACKGROUND: Current treatments for hypercalcemia caused by lung cell carcinomas producing parathyroid hormone-related peptide (PTH-rp) have limited efficacy, probably because of their lack of effect on PTH-rp secretion. In this case study we explored the efficacy of the calcimimetic cinacalcet as su

  5. Central Cell-Derived Peptides Regulate Early Embryo Patterning in Flowering Plants

    NARCIS (Netherlands)

    Costa, L.M.; Marshall, E.; Tesfaye, M.; Silverstein, K.A.T.; Mori, M.; Umetsu, Y.; Otterbach, S.L.; Papareddy, R.; Dickinson, H.G.; Boutilier, K.A.; VandenBosch, K.A.; Ohki, S.; Gutierrez-Marcos, J.F.

    2014-01-01

    Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning i

  6. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    DEFF Research Database (Denmark)

    Hansen, Line; Larsen, Esben Kjær Unmack; Nielsen, Erik Holm;

    2013-01-01

    patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared...

  7. Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters.

    Directory of Open Access Journals (Sweden)

    Matthew D Whim

    Full Text Available BACKGROUND: In addition to polypeptide hormones, pancreatic endocrine cells synthesize a variety of bioactive molecules including classical transmitters and neuropeptides. While these co-transmitters are thought to play a role in regulating hormone release little is known about how their secretion is regulated. Here I investigate the synthesis and release of neuropeptide Y from pancreatic beta cells. METHODOLOGY/PRINCIPAL FINDINGS: NPY appears to be an authentic co-transmitter in neonatal, but not adult, beta cells because (1 early in mouse post-natal development, many beta cells are NPY-immunoreactive whereas no staining is observed in beta cells from NPY knockout mice; (2 GFP-expressing islet cells from an NPY(GFP transgenic mouse are insulin-ir; (3 single cell RT-PCR experiments confirm that the NPY(GFP cells contain insulin mRNA, a marker of beta cells. The NPY-immunoreactivity previously reported in alpha and delta cells is therefore likely to be due to the presence of NPY-related peptides. INS-1 cells, a beta cell line, are also NPY-ir and contain NPY mRNA. Using the FMRFamide tagging technique, NPY secretion was monitored from INS-1 beta cells with high temporal resolution. Peptide release was evoked by brief depolarizations and was potentiated by activators of adenylate cyclase and protein kinase A. Following a transient depolarization, NPY-containing dense core granules fused with the cell membrane and discharged their contents within a few milliseconds. CONCLUSIONS: These results indicate that after birth, NPY expression in pancreatic islets is restricted to neonatal beta cells. The presence of NPY suggests that peptide co-transmitters could mediate rapid paracrine or autocrine signaling within the endocrine pancreas. The FMRFamide tagging technique may be useful in studying the release of other putative islet co-transmitters in real time.

  8. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    Science.gov (United States)

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  9. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    Science.gov (United States)

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. PMID:25621616

  10. Effect of chirality and length on the penetrability of single-walled carbon nanotubes into lipid bilayer cell membranes.

    Science.gov (United States)

    Skandani, A Alipour; Zeineldin, R; Al-Haik, M

    2012-05-22

    The ability of carbon nanotubes to enter the cell membrane acting as drug-delivery vehicles has yielded a plethora of experimental investigations, mostly with inconclusive results because of the wide spectra of carbon nanotube structures. Because of the virtual impossibility of synthesizing CNTs with distinct chirality, we report a parametric study on the use of molecular dynamics to provide better insight into the effect of the carbon nanotube chirality and the aspect ratio on the interaction with a lipid bilayer membrane. The simulation results indicated that a single-walled carbon nanotube utilizes different time-evolving mechanisms to facilitate their internalization within the membrane. These mechanisms comprise both penetration and endocytosis. It was observed that carbon nanotubes with higher aspect ratios penetrate the membrane faster whereas shorter nanotubes undergo significant rotation during the final stages of endocytosis. Furthermore, nanotubes with lower chiral indices developed significant adhesion with the membrane. This adhesion is hypothesized to consume some of the carbon nanotube energy, thus resulting in longer times for the nanotube to translocate through the membrane.

  11. Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion.

    Science.gov (United States)

    Gabriel, Matthias; Niederer, Kerstin; Frey, Holger

    2016-01-01

    Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface. This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period. PMID:27584937

  12. Perlecan domain IV peptide stimulates salivary gland cell assembly in vitro.

    Science.gov (United States)

    Pradhan, Swati; Zhang, Chu; Jia, Xinqiao; Carson, Daniel D; Witt, Robert; Farach-Carson, Mary C

    2009-11-01

    Treatment of xerostomia would benefit from development of a functional implantable artificial salivary gland. Salivary gland tissue from surgical patients was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Ductal and acinar cells were identified in tissue and cultured cells from dispersed tissue. High levels of laminin and perlecan/HSPG2 (heparan sulfate proteoglycan 2) were noted in basement membranes, and perlecan also was secreted and organized by cultured acinar populations, which formed lobular structures that mimicked intact glands when cultured on Matrigel or a bioactive peptide derived from domain IV of perlecan. On either matrix, large acini-like lobular structures grew and formed connections between the lobes. alpha-Amylase secretion was confirmed by staining and activity assay. Biomarkers, including tight junction protein E-cadherin and water channel protein aquaporin 5 found in tissue, were expressed in cultured acinar cells. Cells cultured on Matrigel or domain IV of perlecan peptide organized stress fibers and activated focal adhesion kinase. We report a novel technique to isolate acinar cells from human salivary gland and identify a human peptide sequence in perlecan that triggers differentiation of salivary gland cells into self-assembling acini-like structures that express essential biomarkers and which secrete alpha-amylase.

  13. Nanoparticle-mediated delivery of the antimicrobial peptide plectasin against Staphylococcus aureus in infected epithelial cells

    DEFF Research Database (Denmark)

    Water, Jorrit Jeroen; Smart, Simon; Franzyk, Henrik;

    2015-01-01

    improved efficacy as compared to non-encapsulated plectasin, while the eukaryotic cell viability was unaffected at the assayed concentrations. Further, the subcellular localization of the nanoparticles was assessed in different relevant cell lines. The nanoparticles were distributed in punctuate patterns......A number of pathogenic bacterial strains, such as Staphylococcus aureus, are difficult to kill with conventional antibiotics due to intracellular persistence in host airway epithelium. Designing drug delivery systems to deliver potent antimicrobial peptides (AMPs) intracellularly to the airway...

  14. Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial Cell Activation in Collagen Scaffolds

    OpenAIRE

    Chan, Tania R.; Stahl, Patrick J.; Yu, S. Michael

    2011-01-01

    Long term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial cell (EC) differentiation and vasculature formation are dependent on spatio-temporal cues in the extracellular matrix that dynamically interact with cells, a process difficult to reproduce in artificial systems. Here we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF) and can be used to encode spatially controlled angiogenic signa...

  15. T-Cell Recognition of Mycobacterial GroES Peptides in Thai Leprosy Patients and Contacts

    OpenAIRE

    Chua-Intra, Boosbun; Peerapakorn, Somchai; Davey, Nick; Jurcevic, Stipo; Busson, Marc; Vordermeier, H. Martin; Pirayavaraporn, Charoon; Ivanyi, Juraj

    1998-01-01

    We report here the mapping of T-cell-stimulatory determinants of the GroES 10-kDa heat shock protein homologues from Mycobacterium leprae and Mycobacterium tuberculosis, which are known as major immunogens in mycobacterial infections. Peripheral blood mononuclear cells (PBMC) from treated tuberculoid leprosy or lepromatous leprosy patients and from healthy household or hospital staff contacts of the patients were cultured with 20 16-mer peptides covering the entire sequences of both M. leprae...

  16. Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell

    OpenAIRE

    1984-01-01

    We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C- peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunore...

  17. Improving therapeutic HPV peptide-based vaccine potency by enhancing CD4+ T help and dendritic cell activation

    Directory of Open Access Journals (Sweden)

    Hung Chien-Fu

    2010-11-01

    Full Text Available Abstract Background Effective vaccination against human papillomavirus (HPV represents an opportunity to control cervical cancer. Peptide-based vaccines targeting HPV E6 and/or E7 antigens while safe, will most likely require additional strategies to enhance the vaccine potency. Methods We tested the HPV-16 E7 peptide-based vaccine in combination with a strategy to enhance CD4+ T help using a Pan HLA-DR epitope (PADRE peptide and a strategy to enhance dendritic cell activation using the toll-like receptor 3 ligand, poly(I:C. Results We observed that mice vaccinated with E7 peptide-based vaccine in combination with PADRE peptide and poly(I:C generated better E7-specific CD8+ T cell immune responses as well as significantly improved therapeutic anti-tumor effects against TC-1 tumors compared to E7 peptide-based vaccine with either PADRE peptide or poly(I:C alone. Furthermore, we found that intratumoral vaccination with the E7 peptide in conjunction with PADRE peptide and poly(I:C generates a significantly higher frequency of E7-specific CD8+ T cells as well as better survival compared to subcutaneous vaccination with the same regimen in treated mice. Conclusions The combination of PADRE peptide and poly(I:C with antigenic peptide is capable of generating potent antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated mice. Our study has significant clinical implications for peptide-based vaccination.

  18. Effect of IKVAV Peptide Nanofiber on Proliferation,Adhesion and Differentiation into Neurocytes of Bone Marrow Stromal Cells

    Institute of Scientific and Technical Information of China (English)

    吴斌; 郑启新; 吴永超; 郭晓东; 邹枕玮

    2010-01-01

    This study examined the effect of IKVAV peptide nanofiber on proliferation,adhesion and differentiation into neurocytes of bone marrow stromal cells(BMSCs).IKVAV Peptide-amphiphile was synthesized and purified.Then,hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro.The resultant samples was observed under transmission electron microscope.BMSCs were cultured with IKVAV peptide nanofiber.The effect of IKVAV nanofiber on the proliferation,ad...

  19. How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

    Science.gov (United States)

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

    2012-02-01

    In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

  20. Selenocysteine containing analogues of Atx1-based peptides protect cells from copper ion toxicity.

    Science.gov (United States)

    Shoshan, Michal S; Lehman, Yonat; Goch, Wojciech; Bal, Wojciech; Tshuva, Edit Y; Metanis, Norman

    2016-08-01

    Seleno-substituted model peptides of copper metallochaperone proteins were analyzed for the metal affinity and in vitro anti-oxidative reactivity. An acyclic MTCXXC (X is any amino acid) reference peptide previously analyzed as a potent inhibitor of ROS production underwent substitution of the cysteine residues with selenocysteine to give two singly substituted derivatives C3U and C6U and the doubly substituted analogue C3U/C6U. Presumably due to the softer nature of Se vs. S, all selenocysteine containing peptides demonstrated high affinity to Cu(i), higher than that of the reference peptide, and in the same order of magnitude as that measured for the native protein, Atox1. A stronger impact of residue 3 confirmed previous findings on its more dominant role in metal coordination. In vitro studies on the HT-29 human colon cancer cell line, MEF mice embryonic fibroblasts, and MEF with the knocked-out Atox1 gene (Atox1-/-) consistently identified C3U/C6U as the most potent inhibitor of ROS cellular production based on the 2',7'-dichlorodihydrofluorescin diacetate (H2DCF-DA) assay, also in comparison with known drugs employed in the clinic for Wilson's disease. The selenocysteine containing peptides are thus promising drug candidates for chelation therapy of Wilson's disease and related conditions relevant to excessive copper levels. PMID:27349676

  1. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells

    International Nuclear Information System (INIS)

    Gastrin-releasing peptide (GRP) labeled with 125I at tyrosine-15 (125I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. 125I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells

  2. Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

    Science.gov (United States)

    McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

    2014-03-01

    Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

  3. Peptide p277 of HSP60 signals T cells: inhibition of inflammatory chemotaxis.

    Science.gov (United States)

    Nussbaum, Gabriel; Zanin-Zhorov, Alexandra; Quintana, Francisco; Lider, Ofer; Cohen, Irun R

    2006-10-01

    Peptide p277 is a 24-amino acid fragment of the heat shock protein 60 molecule, first discovered to be an antigen for diabetogenic T-cell clones in non-obese diabetic (NOD) mice. Therapeutic vaccination with p277 can arrest the spontaneous diabetogenic process both in NOD mice and in humans associated with a T(h)1 to T(h)2 cytokine shift specific for the autoimmune T cells. We now report that p277 can directly signal human T cells via innate toll-like receptor (TLR)-2, leading to up-regulation of integrin-mediated adhesion to fibronectin, and inhibition of chemotaxis to the chemokine SDF-1alpha in vitro. Resting CD45RA(+) T cells responded to lower concentrations of p277 than resting CD45RO(+) T cells, but activation of CD45RO(+) T cells greatly increased their sensitivity to p277. Mouse T cells, but not macrophages, were also sensitive to the innate effects of peptide p277, and adoptive transfer of diabetes by splenic T cells from NOD mice could be inhibited by p277 treatment before transfer. Thus, T cells do respond innately to p277, and signaling by soluble p277 through TLR2 could contribute to the treatment of type 1 diabetes; p277 may stop the destruction of beta cells by signaling in concert both innate and adaptive receptors on T cells. PMID:16893923

  4. T cell responses against microsatellite instability-induced frameshift peptides and influence of regulatory T cells in colorectal cancer.

    Science.gov (United States)

    Bauer, Kathrin; Nelius, Nina; Reuschenbach, Miriam; Koch, Moritz; Weitz, Jürgen; Steinert, Gunnar; Kopitz, Jürgen; Beckhove, Philipp; Tariverdian, Mirjam; von Knebel Doeberitz, Magnus; Kloor, Matthias

    2013-01-01

    High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.

  5. Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Naoto, E-mail: nemoto@fms.saitama-u.ac.jp [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844 (Japan); Tsutsui, Chihiro [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Yamaguchi, Junichi [Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Applied Gene Technology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Ueno, Shingo [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Machida, Masayuki [Applied Gene Technology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Kobayashi, Toshikatsu [Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844 (Japan); Sakai, Takafumi [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

  6. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

    Science.gov (United States)

    Ma, Gary S.; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K.; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L.; Ghosh, Pradipta

    2015-01-01

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions. PMID:25926659

  7. A role of TDIF peptide signaling in vascular cell differentiation is conserved among euphyllophytes

    Directory of Open Access Journals (Sweden)

    Yuki eHirakawa

    2015-11-01

    Full Text Available Peptide signals mediate a variety of cell-to-cell communication crucial for plant growth and development. During Arabidopsis thaliana vascular development, a CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related family peptide hormone, TDIF (tracheary element differentiation inhibitory factor, regulates procambial cell fate by its inhibitory activity on xylem differentiation. To address if this activity is conserved among vascular plants, we performed comparative analyses of TDIF signaling in non-flowering vascular plants (gymnosperms, monilophytes and lycophytes. We identified orthologs of TDIF/CLE as well as its receptor TDR/PXY (TDIF RECEPTOR/PHLOEM INTERCALATED WITH XYLEM in Ginkgo biloba, Adiantum aethiopicum and Selaginella kraussiana by RACE-PCR. The predicted TDIF peptide sequences in seed plants and monilophytes were identical to that of A. thaliana TDIF. We examined the effects of exogenous CLE peptide-motif sequences of TDIF in these species. We found that liquid culturing of dissected leaves or shoots was useful for examining TDIF activity during vascular development. TDIF treatment suppressed xylem/tracheary element differentiation of procambial cells in G. bioloba and A. aethiopicum leaves. In contrast, neither TDIF nor putative endogenous TDIF inhibited xylem differentiation in developing shoots and rhizophores of S. kraussiana. These data suggest that activity of TDIF in vascular development is conserved among extant euphyllophytes. In addition to the conserved function, via liquid culturing of its bulbils, we found a novel inhibitory activity on root growth in the monilophyte Asplenium x lucrosum suggesting lineage-specific co-option of peptide signaling occurred during the evolution of vascular plant organs.

  8. Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides.

    Science.gov (United States)

    Hardy, Melinda Y; Tye-Din, Jason A; Stewart, Jessica A; Schmitz, Frederike; Dudek, Nadine L; Hanchapola, Iresha; Purcell, Anthony W; Anderson, Robert P

    2015-01-01

    Celiac disease (CD) is a common CD4(+) T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo. We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5(+) CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye. Avenin-specific responses were observed in 6/73 HLA-DQ2.5(+) CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability. Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.

  9. Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Yuran Xie

    2016-10-01

    Full Text Available The use of small interference RNA (siRNA to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide conjugated to cationic polymer branched polyethylenimine (bPEI, optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299 and low TfR expression (A549 and H460. The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches.

  10. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

    Science.gov (United States)

    Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

    2015-06-23

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

  11. Ultra-small lipid nanoparticles promote the penetration of coenzyme Q10 in skin cells and counteract oxidative stress.

    Science.gov (United States)

    Lohan, Silke B; Bauersachs, Sonja; Ahlberg, Sebastian; Baisaeng, Nuttakorn; Keck, Cornelia M; Müller, Rainer H; Witte, Ellen; Wolk, Kerstin; Hackbarth, Steffen; Röder, Beate; Lademann, Jürgen; Meinke, Martina C

    2015-01-01

    UV irradiation leads to the formation of reactive oxygen species (ROS). An imbalance between the antioxidant system and ROS can lead to cell damage, premature skin aging or skin cancer. To counteract these processes, antioxidants such as coenzyme Q10 (CoQ10) are contained in many cosmetics. To improve and optimize cell/tissue penetration properties of the lipophilic CoQ10, ultra-small lipid nanoparticles (usNLC) were developed. The antioxidant effectiveness of CoQ10-loaded usNLC compared to conventional nanocarriers was investigated in the human keratinocyte cell line HaCaT. Using confocal laser scanning microscopy investigations of the carriers additionally loaded with nile red showed a clear uptake into cells and their distribution within the cytoplasm. By use of the XTT cell viability test, CoQ10 concentrations of 10-50 μg/ml were shown to be non-toxic, and the antioxidant potential of 10 μg/ml CoQ10 loaded usNLC in the HaCaT cells was analyzed via electron paramagnetic resonance spectroscopy after cellular exposure to UVA (1J/cm(2)) and UVB (18 mJ/cm(2)) irradiation. In comparison with the CoQ10-loaded conventional carriers, usNLC-CoQ10 demonstrated the strongest reduction of the radical formation; reaching up to 23% compared to control cells without nanocarrier treatment. Therefore, usNLC-CoQ10 are very suitable to increase the antioxidant potential of skin.

  12. Tuning the anticancer activity of a novel pro-apoptotic peptide using gold nanoparticle platforms

    Science.gov (United States)

    Akrami, Mohammad; Balalaie, Saeed; Hosseinkhani, Saman; Alipour, Mohsen; Salehi, Fahimeh; Bahador, Abbas; Haririan, Ismaeil

    2016-01-01

    Pro-apoptotic peptides induce intrinsic apoptosis pathway in cancer cells. However, poor cellular penetration of the peptides is often associated with limited therapeutic efficacy. In this report, a series of peptide-gold nanoparticle platforms were developed to evaluate the anticancer activity of a novel alpha-lipoic acid-peptide conjugate, LA-WKRAKLAK, with respect to size and shape of nanoparticles. Gold nanoparticles (AuNPs) were found to enhance cell internalization as well as anticancer activity of the peptide conjugates. The smaller nanospheres showed a higher cytotoxicity, morphological change and cellular uptake compared to larger nanospheres and nanorods, whereas nanorods showed more hemolytic activity compared to nanospheres. The findings suggested that the anticancer and biological effects of the peptides induced by intrinsic apoptotic pathway were tuned by peptide-functionalized gold nanoparticles (P-AuNPs) as a function of their size and shape. PMID:27491007

  13. Gliadin peptides induce tissue transglutaminase activation and ER-stress through Ca2+ mobilization in Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Ivana Caputo

    Full Text Available BACKGROUND: Celiac disease (CD is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+ homeostasis and tTG activity. METHODS/PRINCIPAL FINDINGS: We studied Ca(2+ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+ from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+ from the endoplasmic reticulum (ER and mitochondria, whereas peptide 57-68 mobilized Ca(2+ only from mitochondria. We also found that gliadin peptide-induced Ca(2+ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes. CONCLUSIONS: By inducing Ca(2+ mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.

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

    International Nuclear Information System (INIS)

    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

  15. Milk peptides increase iron dialyzability in water but do not affect DMT-1 expression in Caco-2 cells.

    Science.gov (United States)

    Argyri, Konstantina; Tako, Elad; Miller, Dennis D; Glahn, Raymond P; Komaitis, Michael; Kapsokefalou, Maria

    2009-02-25

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. The objectives of this study were to investigate whether these fractions (a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells and (b) enhance iron dialyzability when added in meals. Two milk peptide fractions that solubilize iron were isolated by Sephadex G-25 gel filtration of a milk digest. These peptide fractions did not affect relative gene expression of DMT-1 when incubated with Caco-2 cells for 2 or 48 h. Dialyzability was measured after in vitro simulated gastric and pancreatic digestion. Both peptide fractions enhanced the dialyzability of iron from ferric chloride added to PIPES buffer, but had no effect on dialyzability from milk or a vegetable or fruit meal after in vitro simulated gastric and pancreatic digestion. However, dialyzability from milk was enhanced by the addition of a more concentrated lyophilized peptide fraction.

  16. Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel

    Science.gov (United States)

    Castillo Diaz, Luis A; Elsawy, Mohamed; Saiani, Alberto; Gough, Julie E; Miller, Aline F

    2016-01-01

    An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone. PMID:27493714

  17. A High-Adhesive Lysine-Cyclic RGD Peptide Designed for Selective Cell Retention Technology.

    Science.gov (United States)

    Luo, Keyu; Mei, Tieniu; Li, Zhiqiang; Deng, Moyuan; Zhang, Zehua; Hou, Tianyong; Dong, Shiwu; Xie, Zhao; Xu, Jianzhong; Luo, Fei

    2016-06-01

    Cell adhesion is an important property of biomaterials used in selective cell retention (SCR) technology, which fabricates bone grafts rapidly in clinical settings. This could be improved by physical and biologic manipulations. To facilitate retention of the cells on the scaffold, especially osteoprogenitors from bone marrow in the convenient SCR procedure, a lysine-cyclic RGD (LcRGD) peptide was here designed to coordinate positively charged amino acids and the RGD sequence to enhance the adhesion performance of the scaffold. Demineralized bone matrix (DBM) is an important therapeutic resource, but its cell adhesion ability and osteoinductive capacity are low because of its processing. These capabilities can be increased to enhance the performance of DBM when used in SCR technology. Here, LcRGD peptide was used to modify DBM and produce a DBM/LcRGD composite. This composite exhibited enhanced adhesion performance on cultured human bone marrow-derived mesenchymal stem cells and retained more osteoprogenitors from bone marrow than other materials did. The DBM/LcRGD composite displayed a preferable osteoinduction in vitro and osteogenic capacity in vivo. Thus, LcRGD peptide as a commendable modifier of DBM applied in SCR technology can improve bone transplantation. PMID:27154386

  18. Peptide functionalized polyhydroxyalkanoate nanofibrous scaffolds enhance Schwann cells activity

    NARCIS (Netherlands)

    Masaeli, E.; Wieringa, P.A.; Morshed, M.; Nasr-Esfahani, M.H.; Sadri, S.; Blitterswijk, van C.A.; Moroni, L.

    2014-01-01

    Interactions between Schwann cells (SCs) and scaffolds are important for tissue development during nerve regeneration, because SCs physiologically assist in directing the growth of regenerating axons. In this study, we prepared electrospun scaffolds combining poly (3-hydroxybutyrate) (PHB) and poly

  19. Advantages of RGD peptides for directing cell association with biomaterials

    OpenAIRE

    Bellis, Susan L.

    2011-01-01

    Despite many years of in vitro research confirming the effectiveness of RGD in promoting cell attachment to a wide variety of biomaterials, animal studies evaluating tissue responses to implanted RGD-functionalized substrates have yielded more variable results The goals of this report are to present some of the reasons why cell culture studies may not always reliably predict in vivo responses, and more importantly, to highlight potential applications that may benefit from the use of RGD pepti...

  20. Design of Decorated Self-Assembling Peptide Hydrogels as Architecture for Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Annj Zamuner

    2016-08-01

    Full Text Available Hydrogels from self-assembling ionic complementary peptides have been receiving a lot of interest from the scientific community as mimetic of the extracellular matrix that can offer three-dimensional supports for cell growth or can become vehicles for the delivery of stem cells, drugs or bioactive proteins. In order to develop a 3D “architecture” for mesenchymal stem cells, we propose the introduction in the hydrogel of conjugates obtained by chemoselective ligation between a ionic-complementary self-assembling peptide (called EAK and three different bioactive molecules: an adhesive sequence with 4 Glycine-Arginine-Glycine-Aspartic Acid-Serine-Proline (GRGDSP motifs per chain, an adhesive peptide mapped on h-Vitronectin and the growth factor Insulin-like Growth Factor-1 (IGF-1. The mesenchymal stem cell adhesion assays showed a significant increase in adhesion and proliferation for the hydrogels decorated with each of the synthesized conjugates; moreover, such functionalized 3D hydrogels support cell spreading and elongation, validating the use of this class of self-assembly peptides-based material as very promising 3D model scaffolds for cell cultures, at variance of the less realistic 2D ones. Furthermore, small amplitude oscillatory shear tests showed that the presence of IGF-1-conjugate did not alter significantly the viscoelastic properties of the hydrogels even though differences were observed in the nanoscale structure of the scaffolds obtained by changing their composition, ranging from long, well-defined fibers for conjugates with adhesion sequences to the compact and dense film for the IGF-1-conjugate.

  1. Membrane interaction and secondary structure of de novo designed arginine-and tryptophan peptides with dual function

    KAUST Repository

    Rydberg, Hanna A.

    2012-10-01

    Cell-penetrating peptides and antimicrobial peptides are two classes of positively charged membrane active peptides with several properties in common. The challenge is to combine knowledge about the membrane interaction mechanisms and structural properties of the two classes to design peptides with membrane-specific actions, useful either as transporters of cargo or as antibacterial substances. Membrane active peptides are commonly rich in arginine and tryptophan. We have previously designed a series of arg/trp peptides and investigated how the position and number of tryptophans affect cellular uptake. Here we explore the antimicrobial properties and the interaction with lipid model membranes of these peptides, using minimal inhibitory concentrations assay (MIC), circular dichroism (CD) and linear dichroism (LD). The results show that the arg/trp peptides inhibit the growth of the two gram positive strains Staphylococcus aureus and Staphylococcus pyogenes, with some individual variations depending on the position of the tryptophans. No inhibition of the gram negative strains Proteus mirabilis or Pseudomonas aeruginosa was noticed. CD indicated that when bound to lipid vesicles one of the peptides forms an α-helical like structure, whereas the other five exhibited rather random coiled structures. LD indicated that all six peptides were somehow aligned parallel with the membrane surface. Our results do not reveal any obvious connection between membrane interaction and antimicrobial effect for the studied peptides. By contrast cell-penetrating properties can be coupled to both the secondary structure and the degree of order of the peptides. © 2012 Elsevier Inc.

  2. The effect of beta-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells

    DEFF Research Database (Denmark)

    Sorensen, M; Steenberg, B; Knipp, G T;

    1997-01-01

    PURPOSE: To investigate the effects of the beta-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). METHODS: The effective...... permeability coefficients (Peff) of the model peptides were determined across BBMEC monolayers. The dimensions of the aqueous pores in the tight junctions (TJs) of the BBMEC monolayers were determined using a series of hydrophilic permeants. This value and the molecular radius of each peptide were used...... to calculate the theoretical paracellular (PP*) and transcellular (PT*) permeability coefficients for each peptide. RESULTS: A comparison of the theoretical PP* values with the observed Peff values was made for a series of model peptides. For the most hydrophobic peptides (Ac-PheProXaaIle-NH2 and Ac...

  3. Analysis of Swine Leukocyte Antigen Peptide Binding Profiles and the Identification of T cell Epitopes by Tetramer Staining

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers

    within a species makes immune escape almost impossible for any intruding pathogen. Characterization of the SLA class I and class II gene products and their peptide binding capacity defines the T cell epitopes of any given pathogen proteome. To date the analysis of MHC peptide interactions, strength...... class I peptide binding characteristics in relation to immune responses to vaccination or infection. Applying proven technologies to newly produced, recombinant swine leukocyte antigen (SLA) class I proteins yielded a body of data for peptide:SLA:β2m (pSLA) complex affinity and stability. Mapping...... the SLA proteins for their peptide binding requirements along with the identification of their cognate virally derived peptides, made it possible to explore the nature of the SLA proteins and the roles they play in establishing adaptive immunity. The development of SLA tetramers, enabled investigation...

  4. Peptide-conjugated hapten groups are the major antigenic determinants for trinitrophenyl-specific cytotoxic T cells.

    Science.gov (United States)

    von Bonin, A; Ortmann, B; Martin, S; Weltzien, H U

    1992-08-01

    Several trinitrophenyl (TNP)-specific mouse cytotoxic T cell (CTL) clones recognize TNP-conjugated peptides in association with class I MHC molecules ('hapten-peptide determinants'). However, cell modification with trinitrobenzene sulfonic acid (TNBS) also leads to the formation of TNP determinants covalently attached to MHC molecules ('altered self'). To determine the importance of 'peptide' versus 'altered self' determinants, we used the mutant cell line RMA-S which expresses peptide-free ('empty') Kb and Db molecules at 26 degrees C. Additionally, we stabilized Kb molecules on RMA-S cells at 37 degrees C using the Kb binding heptapeptide N53-59 derived from the vesicular stomatitis virus nucleoprotein. Lacking lysine, this peptide remains unmodified by TNBS and, therefore, only allows the formation of 'altered self' TNP determinants on occupied Kb molecules. RMA-S targets, pretreated or untreated with N53-59, upon TNBS modification were only lysed poorly or not at all by four different TNP-specific CTL. In contrast, all of these clones efficiently lysed TNBS-treated, unmutated RMA cells, and three of them strongly reacted with RMA or RMA-S cells in the presence of tryptic TNP-BSA peptides. Moreover, the clone unreactive for TNP-BSA peptides also recognized TNP self-peptides extracted from TNBS-treated syngeneic spleen cells. Taken together, these data clearly show that TNP residues linked to MHC via associated peptides but not by covalent bondage represent the dominant antigenic epitopes for class I MHC-restricted, hapten-specific T cells. PMID:1384686

  5. Cell penetrating silica nanoparticles doped with two-photon absorbing fluorophores

    NARCIS (Netherlands)

    Bertazza, Loris; Celotti, Lucia; Fabbrini, Graziano; Loi, Maria Antonietta; Maggini, Michele; Mancin, Fabrizio; Marcuz, Silvia; Menna, Enzo; Muccini, Michele; Tonellato, Umberto

    2006-01-01

    Organosilica nanoparticles, doped with two-photon absorbing distyrylbenzene derivatives, were prepared and studied as cell staining agents. Two dyes were used, bearing either two peripheral dimethylamino groups or one dimethylamino and one cyano group. Due to the internal charge transfer character o

  6. Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas

    DEFF Research Database (Denmark)

    Stryhn, A; Pedersen, L O; Ortiz-Navarrete, V;

    1994-01-01

    A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified...... and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could...... be detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8...

  7. Penetration testing

    OpenAIRE

    Zemaníková, Martina

    2010-01-01

    This work focuses on the practical demonstration of design penetration testing. Testing is carried out based on the order of a particular entity/subject which is at their request anonymous. The work is divided into two parts, theoretical and practical. We will be made in first part familiarize with the process and techniques of testing, as is now used in ethical hacking, then we can find in the end appropriate recommendations 6 how to prevent and fight against it in order to protect the...

  8. Enhanced cell-material interactions through the biofunctionalization of polymeric surfaces with engineered peptides.

    Science.gov (United States)

    Punet, Xavier; Mauchauffé, Rodolphe; Giannotti, Marina I; Rodríguez-Cabello, José C; Sanz, Fausto; Engel, Elisabeth; Mateos-Timoneda, Miguel A; Planell, Josep A

    2013-08-12

    Research on surface modification of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin-like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspecific adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for different functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface-cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more efficient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces. PMID:23805782

  9. Measurement of interaction force between RGD-peptide and Hela cell surface by optical tweezers

    Institute of Scientific and Technical Information of China (English)

    Mincheng Zhong; Guosheng Xue; Jinhua Zhou; Ziqiang Wang; Yinmei Li

    2012-01-01

    Since RGD peptides (R:arginine; G:glycine; D:aspartic acid) are found to promote cell adhesion,they are modified at numerous materials surface for medical applications such as drug delivery and regenerative medicine.Peptide-cell surface interactions play a key role in the above applications.In this letter,we study the adhesion force between the RGD-coated bead and Hela cell surface by optical tweezes.The adhesion is dominated by the binding of α5β1 and RGD-peptide with higher adhesion probability and stronger adhesion strength compared with the adhesion of bare bead and cell surface.The binding force for a single α5β1-GRGDSP pair is determined to be 16.8 pN at a loading rate of 1.5 nN/s.The unstressed off-rate is 1.65 × 10-2 s-1 and the distance of transition state for the rigid binding model is 3.0 nm.

  10. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    Science.gov (United States)

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  11. Molecular Physiology of Glucagon-Like Peptide-1 Insulin Secretagogue Action in Pancreatic β Cells

    OpenAIRE

    Leech, Colin A.; Dzhura, Igor; Chepurny, Oleg G.; Kang, Guoxin; Schwede, Frank; Genieser, Hans-G.; Holz, George G.

    2011-01-01

    Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additio...

  12. The use of nanoscale fluorescence microscopic to decipher cell wall modifications during fungal penetration

    OpenAIRE

    Dorothea eEllinger; Christian eVoigt

    2014-01-01

    Plant diseases are one of the most studied subjects in the field of plant science due to their impact on crop yield and food security. Our increased understanding of plant–pathogen interactions was mainly driven by the development of new techniques that facilitated analyses on a subcellular and molecular level. The development of labeling technologies, which allowed the visualization and localization of cellular structures and proteins in live cell imaging, promoted the use of fluorescence an...

  13. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells.

    Science.gov (United States)

    Crosera, Matteo; Prodi, Andrea; Mauro, Marcella; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Pietro; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Filon, Francesca Larese

    2015-08-07

    Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue(®) and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm(2)) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm(2)). Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10(-4) M (MTT assay), 3.8 × 10(-5) M (AlamarBlue(®) assay), and 7.6 × 10(-4) M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

  14. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells

    Directory of Open Access Journals (Sweden)

    Matteo Crosera

    2015-08-01

    Full Text Available Titanium dioxide nanoparticles (TiO2NPs suspensions (concentration 1.0 g/L in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue® and propidium iodide, PI, uptake assays was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm2 while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm2. Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10−4 M (MTT assay, 3.8 × 10−5 M (AlamarBlue® assay, and 7.6 × 10−4 M (PI uptake, index of a necrotic cell death. Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

  15. Comparison of Franz cells and microdialysis for assessing salicylic acid penetration through human skin.

    Science.gov (United States)

    Leveque, N; Makki, S; Hadgraft, J; Humbert, Ph

    2004-01-28

    The purpose of the present study was to compare Franz cells (FC) and microdialysis (MD) for monitoring the skin absorption of salicylic acid (SA). The influence of pH on SA flux was also assessed by these two techniques. Excised abdominal human skin was used in the experiments. SA was dissolved in phosphate buffer solutions of pH 2, 5 and 7 (2 mg/ml). SA concentrations in the receptor FC solutions and in MD samples were assessed by high performance liquid chromatography (HPLC). The results demonstrate that the flux of SA decreased with increased pH. The profiles permeation determined by Franz cells and microdialysis were similar. However, whatever the pH, the SA flux was higher with microdialysis than with Franz cells. The results showed that SA percutaneous permeation conformed to the pH partition hypothesis. The flux of SA was different when it was determined by the two techniques. The collect of SA, by these two techniques is different. The results of the two techniques are compared and discussed.

  16. Screening and Identification of a Novel Hepatocellular Carcinoma Cell Binding Peptide by Using a Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Xiaohua ZHU; Hua WU; Sha LUO; Zhiqun XIANYU; Dan ZHU

    2008-01-01

    The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide (WH-16) was synthesized. The competitive ELISA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery Carrier in target diagnosis or therapy for hHCC.

  17. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  18. Induction, selection and antibacterial activity of the antibacterial peptides from lepldopteran insect cultured cell lines

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coil DH5α to stimulate the antibacterial peptide followed by antibacterial activity assay,induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment.The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest,and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation.A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 ceils in 16 hours after stimulation.Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus,Escherichia coli K12D31 and Salmonella derby.It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli KI2D31 and Salmonella derby.

  19. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    Directory of Open Access Journals (Sweden)

    Ilona Turek

    2015-09-01

    Full Text Available Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP, AtPNP-A (At2g18660 were assessed using quantitative proteomics employing tandem mass tag (TMT labeling and tandem mass spectrometry (LC–MS/MS. In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014 661 and have been deposited to the ProteomeXchange with identifier PXD001386.

  20. Antibody tumor penetration

    Science.gov (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  1. Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells.

    Science.gov (United States)

    Vacas, Eva; Bajo, Ana M; Schally, Andrew V; Sánchez-Chapado, Manuel; Prieto, Juan C; Carmena, María J

    2012-12-01

    Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H(2)O(2)-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H(2)O(2) in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H(2)O(2)-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC(1,2) receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.

  2. Improvement on light penetrability and microalgae biomass production by periodically pre-harvesting Chlorella vulgaris cells with culture medium recycling.

    Science.gov (United States)

    Huang, Yun; Sun, Yahui; Liao, Qiang; Fu, Qian; Xia, Ao; Zhu, Xun

    2016-09-01

    To improve light penetrability and biomass production in batch cultivation, a cultivation mode that periodically pre-harvesting partial microalgae cells from suspension with culture medium recycling was proposed. By daily pre-harvesting 30% microalgae cells from the suspension, the average light intensity in the photobioreactor (PBR) was enhanced by 27.05-122.06%, resulting in a 46.48% increase in total biomass production than that cultivated in batch cultivation without pre-harvesting under an incident light intensity of 160μmolm(-2)s(-1). Compared with the semi-continuous cultivation with 30% microalgae suspension daily replaced with equivalent volume of fresh medium, nutrients and water input was reduced by 60% in the proposed cultivation mode but with slightly decrease (12.82%) in biomass production. No additional nutrient was replenished when culture medium recycling. Furthermore, higher pre-harvesting ratios (40%, 60%) and lower pre-harvesting frequencies (every 2, 2.5days) were not advantageous for the pre-harvesting cultivation mode. PMID:27289058

  3. Recurrent invasive squamous cell carcinoma of the ocular surface requiring penetrating therapeutic sclerokeratoplasty

    Directory of Open Access Journals (Sweden)

    Mark J. Mannis

    2012-12-01

    Full Text Available Purpose: We review a case of invasive squamous cell carcinoma invading the cornea to discuss optimal management. Methods:  Observational case report with histopathologic analysis. Results: Histopathology demonstrates corneal invasion by the tumor that appears to have been completely excised with a large therapeutic keratoplasty and adjuvant cryotherapy. Conclusions: Successful management of ocular surface squamous neoplasia (OSSN requires removal of identifiably abnormal tissue without disruption of normal protective architecture, careful histopathologic analysis, and the employment of adjuvant therapy at the time of or subsequent to surgical excision.

  4. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells

    OpenAIRE

    Matteo Crosera; Andrea Prodi; Marcella Mauro; Marco Pelin; Chiara Florio; Francesca Bellomo; Gianpiero Adami; Pietro Apostoli; Giuseppe Palma; Massimo Bovenzi; Marco Campanini; Francesca Larese Filon

    2015-01-01

    Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue® and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact an...

  5. Encoding Cell-Instructive Cues to PEG-Based Hydrogels via Triple Helical Peptide Assembly

    OpenAIRE

    Stahl, Patrick J.; Yu, S. Michael

    2012-01-01

    Effective synthetic tissue engineering scaffolds mimic the structure and composition of natural extracellular matrix (ECM) to promote optimal cellular adhesion, proliferation, and differentiation. Among many proteins of the ECM, collagen and fibronectin are known to play a key role in the scaffold’s structural integrity as well as its ability to support cell adhesion. Here, we present photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMP...

  6. Delivery of siRNA Complexed with Palmitoylated α-Peptide/β-Peptoid Cell-Penetrating Peptidomimetics

    DEFF Research Database (Denmark)

    Jing, Xiaona; Foged, Camilla; Martin-Bertelsen, Birte;

    2016-01-01

    for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic...... on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using...

  7. Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers

    Science.gov (United States)

    Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

    2015-01-01

    Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

  8. Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells.

    Science.gov (United States)

    Zhou, Mi; Smith, Andrew M; Das, Apurba K; Hodson, Nigel W; Collins, Richard F; Ulijn, Rein V; Gough, Julie E

    2009-05-01

    We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration. PMID:19201459

  9. Identification of Peptides Inhibiting Adhesion of Monocytes to the Injured Vascular Endothelial Cells through Phage-displaying Screening

    Institute of Scientific and Technical Information of China (English)

    Yu GUO; Jia ZHANG; Ji-Cheng WANG; Feng-Xiang YAN; Bing-Yang ZHU; Hong-Lin HUANG; Duan-Fang LIAO

    2005-01-01

    Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the "adsorption-elution-amplification"procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin- 1 and intercellular adhesion molecule- 1 (ICAM- 1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.

  10. Synthetic peptides derived from the Wilms' tumor 1 protein sensitize human T lymphocytes to recognize chronic myelogenous leukemia cells.

    Science.gov (United States)

    Müller, Ludmila; Knights, Ashley; Pawelec, Graham

    2003-01-01

    The Wilms' tumour 1 (WT1) molecule was screened in silico for the presence of 15-mer sequences predicted to bind HLA-DRB1(*)0401 (www.syfpeithi.de). Two peptides with the highest binding scores were synthesized (WT12e, PQQMGSDVRDLNALL and WT331, NKRYFKLSHLQMHSR). In vitro sensitization experiments using PBMC and the 15-mer peptides yielded peptide-specific responses against both WT12e and WT331 from six of seven healthy donors. Moreover, four of four different primary CML cell preparations were directly recognized by five different T cell lines, as assessed by IFN-gamma release. These responses were to a great extent blocked by anti-DR monoclonal antibody. These results suggest that WT1 peptides can be selected that are immunogenic for class II-restricted T-cell responses to native tumor cells, and indicate that they may find application in active immunotherapy of CML. PMID:12692522

  11. Enhanced stem cell tracking via electrostatically assembled fluorescent SPION-peptide complexes

    International Nuclear Information System (INIS)

    For cellular MRI there is a need to label cells with superparamagnetic iron oxide nanoparticles (SPION) that have multiple imaging moieties that are nontoxic and have increased NMR relaxation properties to improve the detection and tracking of therapeutic cells. Although increases in the relaxation properties of SPION have been accomplished, detection of tagged cells is limited by either poor cell labeling efficiency or low intracellular iron content. A strategy via a complex formation with transfection agents to overcome these obstacles has been reported. In this paper, we report a complex formation between negatively charged fluorescent monodisperse SPION and positively charged peptides and use the complex formation to improve the MR properties of labeled stem cells. As a result, labeled stem cells exhibited a strong fluorescent signal and enhanced T 2*-weighted MR imaging in vitro and in vivo in a flank tumor model.

  12. Enhanced stem cell tracking via electrostatically assembled fluorescent SPION-peptide complexes

    Science.gov (United States)

    Lee, Jae-Ho; Smith, Melissa A.; Liu, Wei; Gold, Eric M.; Lewis, Bobbi; Song, Ho-Taek; Frank, Joseph A.

    2009-09-01

    For cellular MRI there is a need to label cells with superparamagnetic iron oxide nanoparticles (SPION) that have multiple imaging moieties that are nontoxic and have increased NMR relaxation properties to improve the detection and tracking of therapeutic cells. Although increases in the relaxation properties of SPION have been accomplished, detection of tagged cells is limited by either poor cell labeling efficiency or low intracellular iron content. A strategy via a complex formation with transfection agents to overcome these obstacles has been reported. In this paper, we report a complex formation between negatively charged fluorescent monodisperse SPION and positively charged peptides and use the complex formation to improve the MR properties of labeled stem cells. As a result, labeled stem cells exhibited a strong fluorescent signal and enhanced T 2*-weighted MR imaging in vitro and in vivo in a flank tumor model.

  13. Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation

    OpenAIRE

    Kang MJ; Park SH; Kang MH; Park MJ; Choi YW

    2013-01-01

    Myung Joo Kang, Sang Han Park, Mean Hyung Kang, Min Jung Park, Young Wook Choi College of Pharmacy, Chung-Ang University, Seoul, Korea Background: A novel dual ligand–modified liposome, folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier (FP-Lipo), was designed to overcome the nonselectivity of conventional penetrating peptide-tagged nanoparticulates and to provide the advantage of selective targeting of the folic acid receptor, which is frequently overexpressed on ep...

  14. PENETRATION ENHANCEMENT OF MEDICINAL AGENTS

    OpenAIRE

    Sharma Ganesh N.; Sanadya Jyotsana; Kaushik Avinash; Dwivedi Abha

    2012-01-01

    Many current therapeutic agents like antibiotics, ionizable and peptide drugs are impermeable or do not possess the requisite physicochemical properties for efficient transport through outer tissue barrier to attain therapeutic blood level. For this reason the delivery of such drugs through barriers is currently one of the major interests in pharmaceutical research. Penetration enhancers or promoters are agents that have no therapeutic properties of their own but can transport the sorption of...

  15. Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays

    Directory of Open Access Journals (Sweden)

    Evans David

    2008-01-01

    Full Text Available Abstract Background The dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection. Results We present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background. Conclusion The use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

  16. The use of nanoscale fluorescence microscopic to decipher cell wall modifications during fungal penetration

    Directory of Open Access Journals (Sweden)

    Dorothea eEllinger

    2014-06-01

    Full Text Available Plant diseases are one of the most studied subjects in the field of plant science due to their impact on crop yield and food security. Our increased understanding of plant–pathogen interactions was mainly driven by the development of new techniques that facilitated analyses on a subcellular and molecular level. The development of labeling technologies, which allowed the visualization and localization of cellular structures and proteins in live cell imaging, promoted the use of fluorescence and laser-scanning microscopy in the field of plant–pathogen interactions. Recent advances in new microscopic technologies opened their application in plant science and in the investigation of plant diseases. In this regard, in planta Förster/Fluorescence resonance energy transfer has demonstrated to facilitate the measurement of protein-protein interactions within the living tissue, supporting the analysis of regulatory pathways involved in plant immunity and putative host-pathogen interactions on a nanoscale level. Localization microscopy, an emerging, non-invasive microscopic technology, will allow investigations with a nanoscale resolution leading to new possibilities in the understanding of molecular processes.

  17. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    International Nuclear Information System (INIS)

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation

  18. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Himaya, S.W.A. [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Dewapriya, Pradeep [Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Kim, Se-Kwon, E-mail: sknkim@pknu.ac.kr [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of)

    2013-06-15

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation.

  19. Immunological features of T cells induced by human telomerase reverse transcriptase-derived peptides in patients with hepatocellular carcinoma.

    Science.gov (United States)

    Mizukoshi, Eishiro; Nakagawa, Hidetoshi; Kitahara, Masaaki; Yamashita, Tatsuya; Arai, Kuniaki; Sunagozaka, Hajime; Fushimi, Kazumi; Kobayashi, Eiji; Kishi, Hiroyuki; Muraguchi, Atsushi; Kaneko, Shuichi

    2015-08-10

    Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme required for telomere elongation. In this study, we investigated the safety and immunogenicity of an hTERT-derived peptide (hTERT461) as a vaccine and characterized the hTERT-specific T cell responses induced. Fourteen hepatocellular carcinoma (HCC) patients were enrolled in the study. The hTERT-derived peptide was emulsified in incomplete Freund's adjuvant and administered via subcutaneous immunization three times biweekly. The maximum toxicity observed was grade 2 according to the common terminology criteria and mainly consisted of skin reactions at the site of vaccination. The vaccination induced hTERT-specific immunity in 71.4% of patients and 57.1% of patients administered with hTERT461 peptide-specific T cells could prevent HCC recurrence after vaccination. In phenotypic analysis, the post-vaccinated increase in hTERT-specific T cells was due to an increase in cells with the effector memory phenotype, with the potential to produce multiple cytokines. Seven hTERT-specific T cell receptors were obtained from the vaccinated patients, showing their cytotoxic activities to hTERT-derived peptide-bearing cells. In conclusion, the safety and effects of immune boosting by hTERT461 peptide have shown the potential of the peptide to provide clinical benefits in HCC patients. PMID:25982205

  20. Peptide production and secretion in GLUTag, NCI-H716 and STC-1 cells: a comparison to native L-cells

    DEFF Research Database (Denmark)

    Kuhre, Rune Ehrenreich; Albrechtsen, Nicolai Jacob Wewer; Deacon, Carolyn F.;

    2016-01-01

    normally co-localizes with GLP-1 in distal L-cells, was not detected in any of the cell lines. GLUTag and STC-1 cells also expressed vasoactive intestinal peptide, but none expressed pancreatic polypeptide or insulin. GLUTag contained and secreted large amounts of cholecystokinin while NCI-H716 did......GLUTag, NCI-H716 and STC-1 are cell lines that are widely used to study mechanisms underlying secretion of glucagon-like peptide (GLP-1), but the extent to which they resemble native L-cells is unknown. We used validated immunoassays for 14 different hormones to analyze peptide content (lysis...... samples; n=9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n=6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2...

  1. Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

    Institute of Scientific and Technical Information of China (English)

    WENG XiuFang; LIANG ZhiHui; LU XiaoLing; ZHONG MaoHua; LU ShengJun; ZHANG CaiE; DENG Jing; WU XiongWen; GONG FeiLi

    2007-01-01

    The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen.The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±1.01%,P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the

  2. RV-23, a Melittin-Related Peptide with Cell-Selective Antibacterial Activity and High Hemocompatibility.

    Science.gov (United States)

    Zhang, Shi-Kun; Ma, Qian; Li, Su-Bo; Gao, Hong-Wei; Tan, Ying-Xia; Gong, Feng; Ji, Shou-Ping

    2016-06-28

    RV-23 is a melittin-related antibacterial peptide (MRP) with lower cytotoxicity than either melittin or AR-23, another MRP. The aim of this study was to explore the mechanism of RV- 23's antibacterial selectivity and its hemocompatibility. The results showed that all the peptides exhibited lytic activity against Staphylococcus aureus and Escherichia coli, with RV-23 showing the highest potency. Moreover, RV-23 had lower cytotoxicity than melittin or AR-23 at their minimal inhibitory concentration. In addition, CD experiments showed that melittin, RV-23, and AR-23 all had a typical α-helical structure, and RV-23 had the lowest α-helix content. The structural information showed that RV-23 has the lowest hydrophobicity and highest hydrophobic moment. Because hydrophobicity and α-helix content are believed to correlate with hemolysis, the results indicate that the selective lytic activity against bacteria of RV-23 may be due to its low hydrophobicity and α-helicity, which lead to low cytotoxicity without affecting antibacterial activity. Furthermore, RV-23 did not affect the structure and function of blood components such as red blood cells, platelets, albumin, and the blood coagulation system. In conclusion, RV-23 is a cell-selective antibacterial peptide with high hemocompatibility due to its unique structure. PMID:26975766

  3. [Peptide vaccine therapy with TLR-9 agonist for patients with esophageal squamous cell carcinoma].

    Science.gov (United States)

    Katsuda, Masahiro; Iwahashi, Makoto; Matsuda, Kenji; Miyazawa, Motoki; Nakamori, Mikihito; Nakamura, Masaki; Naka, Teiji; Ojima, Toshiyasu; Iida, Takeshi; Yamaue, Hiroki

    2011-11-01

    Patients with advanced carcinoma are thought to have an impaired immune surveillance system. Therefore, the potent helper action is required for the induction of an antitumor immune response in such patients. We evaluated the efficacy of CpG-ODN, which is TLR-9 agonist, as cancer vaccine adjuvant through in vitro experiments. We also conducted a phase I clinical trial for patients with advanced esophageal squamous cell carcinoma (ESCC) using peptide vaccine in combination with CpG-B. In vitro experiments showed that CpG-ODN caused various immune-modifications, suggesting an efficacy of CpG-ODN as peptide vaccine adjuvant. Moreover, the immune monitoring data in phase I clinical trial suggested that CpG-B augmented the generation of antigen-specific T cell responses and innate immunity. These data indicated that the vaccination with cancer-testis antigen derived peptide in combination with CpG-B may be useful as a new immunotherapy for patients with advanced ESCC. PMID:22202246

  4. Inhibitory effects of small molecular peptides from Spirulina (Arthrospira) platensis on cancer cell growth.

    Science.gov (United States)

    Wang, Zhujun; Zhang, Xuewu

    2016-02-01

    In this study, the whole proteins of Spirulina (Arthrospira) platensis were extracted, hydrolysis with three proteases (trypsin, alcalase and papain) was performed, and gel filtration chromatography was employed to separate hydrolysates. Totally, 15 polypeptides were isolated, which showed anti-proliferation activities on five cancer cells (HepG-2, MCF-7, SGC-7901, A549 and HT-29), with the IC50 values between <31.25 and 336.57 μg mL(-1). Moreover, a new peptide YGFVMPRSGLWFR was identified from papain-digested hydrolysates. It also exhibited inhibitory activities on cancer cells, and the best activity was observed on A549 cancer cells (IC50 values 104.05 μg mL(-1)). In other words, these polypeptides exhibited anti-proliferation activities on cancer cells, and low toxicity or stimulatory activity on normal cells, suggesting that they are promising ingredients in food and pharmaceutical applications. PMID:26584028

  5. Development of a market penetration forecasting model for Hydrogen Fuel Cell Vehicles considering infrastructure and cost reduction effects

    International Nuclear Information System (INIS)

    In order to cope with climate change, the development and deployment of Hydrogen Fuel Cell Vehicles (HFCVs) is becoming more important. In this study, we developed a forecasting model for HFCVs based on the generalized Bass diffusion model and a simulation model using system dynamics. Through the developed model, we could forecast that the saturation of HFCVs in Korea can be moved up 12 years compared with the US. A sensitivity analysis on external variables such as price reduction rates of HFCVs and number of hydrogen refueling stations is also conducted. The results of this study can give insights on the effects of external variables on the market penetration of HFCVs, and the developed model can also be applied to other studies in analyzing the diffusion effects of HFCVs. - Highlights: → A forecasting model for HFCVs was developed using the generalized Bass diffusion model. → A simulation model using system dynamics was also developed. → The empirical study shows that the infrastructure is an important factor to the initial purchase. → The results of this study can promote research related to the diffusion of innovation.

  6. Cell surface-expressed moesin-like receptor regulates T cell interactions with tissue components and binds an adhesion-modulating IL-2 peptide generated by elastase.

    Science.gov (United States)

    Ariel, A; Hershkoviz, R; Altbaum-Weiss, I; Ganor, S; Lider, O

    2001-03-01

    The adhesion of leukocytes to the extracellular matrix (ECM) depends on their responses to variations in the chemotactic signals in their milieu, as well as on the functioning of cytoskeletal and context-specific receptors. Ezrin, radixin, and moesin constitute a family of proteins that link the plasma membrane to the actin cytoskeleton. The surface expression of moesin on T cells and its role in cell adhesion has not been fully elucidated. Recently, we found that IL-2 peptides generated by elastase modified the adhesion of activated T cells to ECM ligands. Here, we further examined the adhesion regulatory effects of EFLNRWIT, one of the IL-2 peptides, as well as the existence and putative function of its receptor on T cells. We found that when presented to T cells in the absence of another activator, the EFLNRWIT peptide induced cell adhesion to vessel wall and ECM components. Binding of a radiolabeled peptide to T cells, precipitation with the immobilized peptide, and amino acid sequencing of the precipitated protein revealed that EFLNRWIT exerts its function via a cell surface-expressed moesin-like moiety, whose constitutive expression on T cells was increased after activation. This notion was further supported by our findings that: 1) anti-moesin mAb inhibited the binding of T cells to the immobilized EFLNRWIT peptide, 2) immobilized recombinant moesin bound the IL-2 peptide, and 3) soluble moesin inhibited the EFLNRWIT-induced T cell adhesion to fibronectin. Interestingly, moesin appears to be generally involved in T cell responses to adhesion-regulating signals. Thus, the IL-2 peptide EFLNRWIT appears to exert its modulating capacities via an adhesion-regulating moesin-like receptor. PMID:11207255

  7. HIV-1 Capsid Assembly Inhibitor (CAI Peptide: Structural Preferences and Delivery into Human Embryonic Lung Cells and Lymphocytes

    Directory of Open Access Journals (Sweden)

    Klaus Braun, Martin Frank, Rüdiger Pipkorn, Jennifer Reed, Herbert Spring, Jürgen Debus, Bernd Didinger, Claus-Wilhelm von der Lieth, Manfred Wiessler, Waldemar Waldeck

    2008-01-01

    Full Text Available The Human immunodeficiency 1 derived capsid assembly inhibitor peptide (HIV-1 CAI-peptide is a promising lead candidate for anti-HIV drug development. Its drawback, however, is that it cannot permeate cells directly. Here we report the transport of the pharmacologically active CAI-peptide into human lymphocytes and Human Embryonic Lung cells (HEL using the BioShuttle platform. Generally, the transfer of pharmacologically active substances across membranes, demonstrated by confocal laser scanning microscopy (CLSM, could lead to a loss of function by changing the molecule's structure. Molecular dynamics (MD simulations and circular dichroism (CD studies suggest that the CAI-peptide has an intrinsic capacity to form a helical structure, which seems to be critical for the pharmacological effect as revealed by intensive docking calculations and comparison with control peptides. This coupling of the CAI-peptide to a BioShuttle-molecule additionally improved its solubility. Under the conditions described, the HIV-1 CAI peptide was transported into living cells and could be localized in the vicinity of the mitochondria.

  8. HIV-1 Capsid Assembly Inhibitor (CAI) Peptide: Structural Preferences and Delivery into Human Embryonic Lung Cells and Lymphocytes

    Science.gov (United States)

    Braun, Klaus; Frank, Martin; Pipkorn, Rüdiger; Reed, Jennifer; Spring, Herbert; Debus, Jürgen; Didinger, Bernd; von der Lieth, Claus-Wilhelm; Wiessler, Manfred; Waldeck, Waldemar

    2008-01-01

    The Human immunodeficiency virus 1 derived capsid assembly inhibitor peptide (HIV-1 CAI-peptide) is a promising lead candidate for anti-HIV drug development. Its drawback, however, is that it cannot permeate cells directly. Here we report the transport of the pharmacologically active CAI-peptide into human lymphocytes and Human Embryonic Lung cells (HEL) using the BioShuttle platform. Generally, the transfer of pharmacologically active substances across membranes, demonstrated by confocal laser scanning microscopy (CLSM), could lead to a loss of function by changing the molecule's structure. Molecular dynamics (MD) simulations and circular dichroism (CD) studies suggest that the CAI-peptide has an intrinsic capacity to form a helical structure, which seems to be critical for the pharmacological effect as revealed by intensive docking calculations and comparison with control peptides. This coupling of the CAI-peptide to a BioShuttle-molecule additionally improved its solubility. Under the conditions described, the HIV-1 CAI peptide was transported into living cells and could be localized in the vicinity of the mitochondria. PMID:18695744

  9. Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

    Directory of Open Access Journals (Sweden)

    Liezl Rae Balaoing

    Full Text Available Valve endothelial cells (VEC have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol diacrylate (PEGDA hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF. VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator and thrombotic (VWF, tissue factor, and P-selectin proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In

  10. Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

    Science.gov (United States)

    Balaoing, Liezl Rae; Post, Allison Davis; Lin, Adam Yuh; Tseng, Hubert; Moake, Joel L; Grande-Allen, K Jane

    2015-01-01

    Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion

  11. Models of self-peptide sampling by developing T cells identify candidate mechanisms of thymic selection.

    Directory of Open Access Journals (Sweden)

    Iren Bains

    Full Text Available Conventional and regulatory T cells develop in the thymus where they are exposed to samples of self-peptide MHC (pMHC ligands. This probabilistic process selects for cells within a range of responsiveness that allows the detection of foreign antigen without excessive responses to self. Regulatory T cells are thought to lie at the higher end of the spectrum of acceptable self-reactivity and play a crucial role in the control of autoimmunity and tolerance to innocuous antigens. While many studies have elucidated key elements influencing lineage commitment, we still lack a full understanding of how thymocytes integrate signals obtained by sampling self-peptides to make fate decisions. To address this problem, we apply stochastic models of signal integration by T cells to data from a study quantifying the development of the two lineages using controllable levels of agonist peptide in the thymus. We find two models are able to explain the observations; one in which T cells continually re-assess fate decisions on the basis of multiple summed proximal signals from TCR-pMHC interactions; and another in which TCR sensitivity is modulated over time, such that contact with the same pMHC ligand may lead to divergent outcomes at different stages of development. Neither model requires that T(conv and T(reg are differentially susceptible to deletion or that the two lineages need qualitatively different signals for development, as have been proposed. We find additional support for the variable-sensitivity model, which is able to explain apparently paradoxical observations regarding the effect of partial and strong agonists on T(conv and T(reg development.

  12. Peptide micelle-mediated curcumin delivery for protection of islet β-cells under hypoxia.

    Science.gov (United States)

    Han, Jaesik; Oh, Jungju; Ihm, Sung-Hee; Lee, Minhyung

    2016-08-01

    Islet transplantation is one of many therapeutic approaches for the treatment of diabetes. During transplant procedures, the isolated islets are subjected to hypoxic conditions, and undergo the apoptotic process. Curcumin has a cytoprotective effect, and may therefore be useful for the protection of islets under hypoxia. However, curcumin is hydrophobic, and an efficient curcumin carrier is required for effective treatment. In this study, R3V6 peptide micelles, composed of a 3-arginine stretch and 6-valine stretch, were evaluated as a curcumin carrier to INS-1 insulinoma cells. Curcumin was loaded into R3V6 micelles at a weight ratio of 10:3 (R3V6:curcumin). The size and surface charge of the curcumin-loaded R3V6 micelles (R3V6-curcumin) were approximately 250 nm and 17.49 mV, respectively. R3V6-curcumin delivered curcumin to the INS-1 cells more efficiently than either curcumin alone or a simple mixture of R3V6 and curcumin. MTT assay indicated that under hypoxia, R3V6-curcumin protected INS-1 cells more efficiently than curcumin alone. TUNEL and reactive oxygen species (ROS) assays suggested that R3V6-curcumin reduced INS-1 cell apoptosis under hypoxia. These results demonstrate that R3V6 peptide micelles are an effective carrier of curcumin, and that R3V6-curcumin may improve the viability of pancreatic β-cells in islet transplantation. PMID:26768151

  13. Dendritic-tumor fusion cells derived heat shock protein70-peptide complex has enhanced immunogenicity.

    Science.gov (United States)

    Zhang, Yunfei; Zhang, Yong; Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use.

  14. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer.

    Science.gov (United States)

    Silva, Vera L; Ferreira, Debora; Nobrega, Franklin L; Martins, Ivone M; Kluskens, Leon D; Rodrigues, Ligia R

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line- 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 -CPTASNTSC and 4T1pep2-EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  15. Enteral peptide formulas inhibit radiation induced enteritis and apoptosis in intestinal epithelial cells and suppress the expression and function of Alzheimer's and cell division control gene products

    International Nuclear Information System (INIS)

    Studies have shown that patients receiving enteral peptide formulas prior to irradiation have a significantly reduced incidence of enteritis and express a profound increase in intestinal cellularity. Two conceptual approaches were taken to describe this response. First was the evaluation in changes in programmed intestinal cell death and secondly the evaluation of a gene product controlling cell division cycling. This study provided a relationship between the ratio of cell death to cell formulations. The results indicate that in the canine and murine models, irradiation induces expression of the Alzheimer's gene in intestinal crypt cells, while the incidence of apoptosis in apical cells is significantly increased. The use of peptide enteral formulations suppresses the expression of the Alzheimer's gene in crypt cells, while apoptosis is eliminated in the apical cells of the intestine. Concomitantly, enteral peptide formulations suppress the function of the CK-II gene product in the basal and baso-lateral cells of the intestine. These data indicate that although the mitotic index is significantly reduced in enterocytes, this phenomenon alone is not sufficient to account for the peptide-induced radio-resistance of the intestine. The data also indicate a significant reduction of normal apoptosis in the upper lateral and apical cells of the intestinal villi. Thus, the ratio of cell death to cell replacement is significantly decreased resulting in an increase in villus height and hypertrophy of the apical villus cells. Thus, peptide solutions should be considered as an adjunct treatment both in radio- and chemotherapy

  16. Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloid-β peptide in mouse brain capillary endothelial cells.

    Science.gov (United States)

    Ito, Shingo; Ohtsuki, Sumio; Murata, Sho; Katsukura, Yuki; Suzuki, Hiroya; Funaki, Miho; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-01-01

    Cerebral clearance of amyloid-β peptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

  17. Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles

    Directory of Open Access Journals (Sweden)

    Taguchi Hiroaki

    2011-08-01

    Full Text Available Abstract Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens, carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1 the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2 synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems.

  18. General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

    Science.gov (United States)

    Dathe, Margitta; Meyer, Jana; Beyermann, Michael; Maul, Björn; Hoischen, Christian; Bienert, Michael

    2002-02-01

    Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.

  19. Peptide-beta2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells.

    Science.gov (United States)

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-09-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-beta2-microglobulin-MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide-MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9.2 x 10(8) antigen-specific cytotoxic CD8(+) T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8(+) T cells, which could be used for adoptive T-cell therapy.

  20. Glucagon-like-peptide-1 secretion from canine L-cells is increased by glucose-dependent-insulinotropic peptide but unaffected by glucose

    DEFF Research Database (Denmark)

    Damholt, A B; Buchan, A M; Kofod, Hans

    1998-01-01

    Glucagon-like peptide-1(7-36)amide (GLP-1) is a potent insulinotropic peptide released from the small intestine. To investigate the regulation of GLP-1 secretion, we established a GLP-1 release assay based on primary canine intestinal L-cells. The ileal mucosa was digested with collagenase...... but not by staurosporine. These results indicate that glucose does not directly stimulate canine L-cells. It is more probable that glucose releases GIP from the upper intestine that in turn stimulates GLP-1 secretion. The ability of GIP to stimulate GLP-1 secretion is probably mediated through activation of protein kinase...... dependently stimulated the release of GLP-1 and resulted in a 2-fold increase at 100 nM GIP. This effect was fully inhibited by 10 nM somatostatin. However, neither basal or GIP stimulated GLP-1 secretion were affected by ambient glucose concentrations from 5-25 mM. The receptor-independent secretagogues beta...

  1. Peptide-specific,allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from pep-tide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85)was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±0.01 %, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the

  2. Reiterated Targeting Peptides on the Nanoparticle Surface Significantly Promote Targeted Vascular Endothelial Growth Factor Gene Delivery to Stem Cells.

    Science.gov (United States)

    Wang, Dong-Dong; Yang, Mingying; Zhu, Ye; Mao, Chuanbin

    2015-12-14

    Nonviral gene delivery vectors hold great promise for gene therapy due to the safety concerns with viral vectors. However, the application of nonviral vectors is hindered by their low transfection efficiency. Herein, in order to tackle this challenge, we developed a nonviral vector integrating lipids, sleeping beauty transposon system and 8-mer stem cell targeting peptides for safe and efficient gene delivery to hard-to-transfect mesenchymal stem cells (MSCs). The 8-mer MSC-targeting peptides, when synthetically reiterated in three folds and chemically presented on the surface, significantly promoted the resultant lipid-based nanoparticles (LBNs) to deliver VEGF gene into MSCs with a high transfection efficiency (∼52%) and long-lasting gene expression (for longer than 170 h) when compared to nonreiterated peptides. However, the reiterated stem cell targeting peptides do not enable the highly efficient gene transfer to other control cells. This work suggests that the surface presentation of the reiterated stem cell-targeting peptides on the nonviral vectors is a promising method for improving the efficiency of cell-specific nonviral gene transfection in stem cells. PMID:26588028

  3. The effects of glucagon-like peptide-1 on the beta cell

    DEFF Research Database (Denmark)

    Vilsbøll, Tina

    2009-01-01

    Type 2 diabetes is a progressive disease characterized by insulin resistance and impaired beta-cell function. Treatments that prevent further beta-cell decline are therefore essential for the management of type 2 diabetes. Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is known...... to stimulate glucose-dependent insulin secretion. Furthermore, GLP-1 appears to have multiple positive effects on beta cells. However, GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4), which limits the clinical relevance of GLP-1 for the treatment of type 2 diabetes. Two main classes of GLP-1-based...... therapies have now been developed: DPP-4 inhibitors and GLP-1 receptor agonists. Liraglutide and exenatide are examples of GLP-1 receptor agonists that have been developed to mimic the insulinotropic characteristics of endogenous GLP-1. Both have demonstrated improved beta-cell function in patients...

  4. Expression of HSV-1 ICP0 Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.

  5. Physicochemical properties of peptide-coated microelectrode arrays and their in vitro effects on neuroblast cells.

    Science.gov (United States)

    Ghane-Motlagh, Bahareh; Javanbakht, Taraneh; Shoghi, Fatemeh; Wilkinson, Kevin J; Martel, Richard; Sawan, Mohamad

    2016-11-01

    Silicon micromachined neural electrode arrays, which act as an interface between bioelectronic devices and neural tissues, play an important role in chronic implants, in vivo. The biological compatibility of chronic microelectrode arrays (MEA) is an essential factor that must be taken into account in their design and fabrication. In order to improve biocompatibility of the MEAs, the surface of the electrodes was coated with polyethylene glycol (PEG) and parylene-C, which are biocompatible polymers. An in vitro study was performed to test the capacity of poly-d-lysine (PDL) to improve neural-cell adhesion and proliferation. Increased proliferation of the neuroblast cells on the microelectrodes was observed in the presence of the PDL. The presence of the peptide on the electrode surface was confirmed using Fourier transform infrared spectroscopy and scanning electron microscopy (SEM). The impedance of the electrodes was not changed significantly before and after PDL deposition. Mouse neuroblast cells were seeded and cultured on the PDL coated and uncoated neural MEAs with different tip-coatings such as platinum, molybdenum, gold, sputtered iridium oxide, and carbon nanotubes. The neuroblast cells grew preferentially on and around peptide coated-microelectrode tips, as compared to the uncoated microelectrodes. PMID:27524064

  6. TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

    Science.gov (United States)

    Takada, Kensuke; Van Laethem, Francois; Xing, Yan; Akane, Kazuyuki; Suzuki, Haruhiko; Murata, Shigeo; Tanaka, Keiji; Jameson, Stephen C; Singer, Alfred; Takahama, Yousuke

    2015-10-01

    In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

  7. Peptide hydrogels – versatile matrices for 3D cell culture in cancer medicine

    Directory of Open Access Journals (Sweden)

    Peter eWorthington

    2015-04-01

    Full Text Available Traditional two-dimensional (2D cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell-cell and cell-material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allow for the development of concentration gradients, including oxygen, metabolites and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture are short sequence, self-assembling peptide hydrogels. In addition to the review of recent work towards the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.

  8. Phase I Trial of a CD8+ T-Cell Peptide Epitope-Based Vaccine for Infectious Mononucleosis▿

    OpenAIRE

    Elliott, Suzanne L.; Suhrbier, Andreas; Miles, John J.; Lawrence, Greg; Pye, Stephanie J.; Le, Thuy T.; Rosenstengel, Andrew; Nguyen, Tam; Allworth, Anthony; Burrows, Scott R.; COX, JOHN; Pye, David; Moss, Denis J.; Bharadwaj, Mandvi

    2007-01-01

    A single blind, randomized, placebo-controlled, single-center phase I clinical trial of a CD8+ T-cell peptide epitope vaccine against infectious mononucleosis was conducted with 14 HLA B*0801-positive, Epstein-Barr virus (EBV)-seronegative adults. The vaccine comprised the HLA B*0801-restricted peptide epitope FLRGRAYGL and tetanus toxoid formulated in a water-in-oil adjuvant, Montanide ISA 720. FLRGRAYGL-specific responses were detected in 8/9 peptide-vaccine recipients and 0/4 placebo vacci...

  9. Putative signal peptides of two BURP proteins can direct proteins to their destinations in tobacco cell system.

    Science.gov (United States)

    Tang, Yulin; Ou, Zhonghua; Qiu, Jianbin; Mi, Zilan

    2014-11-01

    Plant-specific BURP family proteins have a diverse subcellular localization with different functions. However, only limited studies have investigated the functions of their different domains. In the present study, the role of the N-terminal putative signal peptide in protein subcellular localization was investigated using a tobacco cell system. The results showed that SALI3-2 was present in vacuoles, whereas AtRD22 was directed to the apoplast. The N-terminal putative signal peptides of both proteins were confirmed to be the essential and critical domains for targeting the proteins to their destinations. We also demonstrate that the expression and accumulation of mGFP in tobacco cells was increased when mGFP was fused to the putative signal peptide of SALI3-2. The findings offer the potential application of this short peptide in protein production in plants. PMID:25048229

  10. Peptide conjugates for chromosomal gene targeting by triplex-forming oligonucleotides

    OpenAIRE

    Rogers, Faye A.; Manoharan, Muthiah; Rabinovitch, Peter; Ward, David C.; Glazer, Peter M.

    2004-01-01

    Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we...

  11. Chromogranin A-derived peptides are involved in innate immunity.

    Science.gov (United States)

    Aslam, R; Atindehou, M; Lavaux, T; Haïkel, Y; Schneider, F; Metz-Boutigue, M-H

    2012-01-01

    New endogenous antimicrobial peptides (AMPs) derived from chromogranin A (CgA) are secreted by nervous, endocrine and immune cells during stress. They display antimicrobial activities by lytic effects at micromolar range using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. These AMPs can also penetrate quickly into neutrophils (without lytic effects), where, similarly to "cell penetrating peptides", they interact with cytoplasmic calmodulin, and induce calcium influx via Store Operated Channels therefore triggering neutrophils activation. Staphylococcus aureus and Salmonella enteritis are bacteria responsible for severe infections. We investigated here the effects of S. aureus and S. enteritis bacterial proteases on CgA-derived peptides and evaluated their antimicrobial activities. We showed that the Glu-C protease produced by S. aureus V8 induces the loss of the AMPs antibacterial activities and produces new antifungal peptides. In addition, four antimicrobial CGA-derived peptides (chromofungin, procatestatin, human/bovine catestatin) are degraded when treated with bacterial supernatants from S. aureus and S. enteritis, whereas, cateslytin, the short active form of catestatin, resists to this degradation. Finally, we demonstrate that several antimicrobial CgA-derived peptides are able to act synergistically with antibiotics against bacteria and fungi indicating their roles in innate defense.

  12. A peptide-mediated targeting gene delivery system for malignant glioma cells

    Directory of Open Access Journals (Sweden)

    Wang C

    2013-09-01

    Full Text Available Chuanwei Wang,1,2,* Liping Ning,3,* Hongwei Wang,1,2,* Zaijun Lu,4 Xingang Li,1,2 Xiaoyong Fan,5 Xuping Wang,6 Yuguang Liu1,2 1Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, People's Republic of China; 2Brain Science Research Institute of Shandong University, Jinan, People's Republic of China; 3Department of Rehabilitation, Provincial Hospital Affiliated to Shandong University, Jinan, People's Republic of China; 4School of Chemistry and Chemical Engineering of Shandong University, Jinan, People's Republic of China; 5Department of Neurosurgery, Shandong Qianfoshan Hospital Affiliated to Shandong University, Jinan, People's Republic of China; 6Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan, People's Republic of China *These authors contributed equally to this work Abstract: Glioblastoma multiforme (GBM is the most common and malignant glioma. Although there has been considerable progress in treatment strategies, the prognosis of many patients with GBM remains poor. In this work, polyethylenimine (PEI and the VTWTPQAWFQWV (VTW peptide were modified and synthesized into GBM-targeting nanoparticles. The transfection efficiency of U-87 (human glioblastoma cells was evaluated using fluorescence microscopy and flow cytometry. Cell internalization was investigated to verify the nanoparticle delivery into the cytoplasm. Results showed that the methods of polymer conjugation and the amount of VTW peptide were important factors to polymer synthesis and transfection. The PEI-VTW20 nanoparticles increased the transfection efficiency significantly. This report describes the use of VTW peptide-based PEI nanoparticles for intracellular gene delivery in a GBM cell-specific manner. Keywords: glioblastoma, polyethylenimine, nanoparticles, drug-delivery systems, gene transfer techniques

  13. 3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds

    Directory of Open Access Journals (Sweden)

    Cunha C

    2011-05-01

    Full Text Available Carla Cunha1,2, Silvia Panseri3,4, Omar Villa1,2, Diego Silva1,2, Fabrizio Gelain1,21Department of Biotechnology and Biosciences, University of Milano-Bicocca; 2Center for Nanomedicine and Tissue Engineering, CNTE – A.O. Ospedale Niguarda Ca' Granda, Milan; 3Laboratory of Biomechanics and Technology Innovation, Rizzoli Orthopaedic Institute, Bologna; 4Laboratory of Nano-Biomagnetism, Institute of Science and Technology for Ceramics, National Research Council, Faenza, ItalyAbstract: Three-dimensional (3D in vitro models of cell culture aim to fill the gap between the standard two-dimensional cell studies and the in vivo environment. Especially for neural tissue regeneration approaches where there is little regenerative capacity, these models are important for mimicking the extracellular matrix in providing support, allowing the natural flow of oxygen, nutrients, and growth factors, and possibly favoring neural cell regrowth. We have previously demonstrated that a new self-assembling nanostructured biomaterial, based on matrigel, was able to support adult neural stem cell (NSC culture. In this study, we developed a new 3D cell culture system that takes advantage of the nano- and microfiber assembling process, under physiologic conditions, of these biomaterials. The assembled scaffold forms an intricate and biologically active matrix that displays specifically designed functional motifs: RGD (Arg-Gly-Asp, BMHP1 (bone marrow homing peptide 1, and BMHP2, for the culture of adult NSCs. These scaffolds were prepared at different concentrations, and microscopic examination of the cell-embedded scaffolds showed that NSCs are viable and they proliferate and differentiate within the nanostructured environment of the scaffold. Such a model has the potential to be tailored to develop ad hoc designed peptides for specific cell lines.Keywords: biomaterials, tissue engineering, 3D in vitro model

  14. A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wu Xiaoyan; Chen Peili [Department of Medicine, University of Chicago, Chicago, Illinois (United States); Sonis, Stephen T. [Division of Oral Medicine, Brigham and Women' s Hospital, Boston, Massachusetts (United States); Biomodels, Watertown, Massachusetts (United States); Lingen, Mark W. [Department of Pathology, University of Chicago, Chicago, Illinois (United States); Berger, Ann [NephRx Corporation, Kalamazoo, Michigan (United States); Toback, F. Gary, E-mail: gtoback@medicine.bsd.uchicago.edu [Department of Medicine, University of Chicago, Chicago, Illinois (United States)

    2012-07-01

    Purpose: No effective agents currently exist to treat oral mucositis (OM) in patients receiving chemoradiation for the treatment of head-and-neck cancer. We identified a novel 21-amino acid peptide derived from antrum mucosal protein-18 that is cytoprotective, mitogenic, and motogenic in tissue culture and animal models of gastrointestinal epithelial cell injury. We examined whether administration of antrum mucosal protein peptide (AMP-p) could protect against and/or speed recovery from OM. Methods and Materials: OM was induced in established hamster models by a single dose of radiation, fractionated radiation, or fractionated radiation together with cisplatin to simulate conventional treatments of head-and-neck cancer. Results: Daily subcutaneous administration of AMP-p reduced the occurrence of ulceration and accelerated mucosal recovery in all three models. A delay in the onset of erythema after irradiation was observed, suggesting that a protective effect exists even before injury to mucosal epithelial cells occurs. To test this hypothesis, the effects of AMP-p on tumor necrosis factor-{alpha}-induced apoptosis were studied in an endothelial cell line (human dermal microvascular endothelial cells) as well as an epithelial cell line (human adult low-calcium, high-temperature keratinocytes; HaCaT) used to model the oral mucosa. AMP-p treatment, either before or after cell monolayers were exposed to tumor necrosis factor-{alpha}, protected against development of apoptosis in both cell types when assessed by annexin V and propidium iodide staining followed by flow cytometry or ligase-mediated polymerase chain reaction. Conclusions: These observations suggest that the ability of AMP-p to attenuate radiation-induced OM could be attributable, at least in part, to its antiapoptotic activity.

  15. Science Letters: Brain natriuretic peptide: A potential indicator of cardiomyogenesis after autologous mesenchymal stem cell transplantation?

    Institute of Scientific and Technical Information of China (English)

    LI Nan; WANG Jian-an

    2006-01-01

    We observed in a pilot study that there was a transient elevation of brain natriuretic peptide (BNP) level shortly after the transplantation in the patient with ischemic heart failure, which is unexplainable by the simultaneous increase of the cardiac output and six-minute walk distance. Similar findings were observed in the phase I trial. We postulated on the basis of the finding of Fukuda in vitro that this transient elevation of BNP level against the improvement of cardiac function and exercise capacity might indicate cardiomyogenesis in patients after mesenchymal stem cell transplantation. Further study is warranted to verify the hypothesis.

  16. The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

    Directory of Open Access Journals (Sweden)

    Rekdal Øystein

    2009-06-01

    Full Text Available Abstract Background Cationic antimicrobial peptides (CAPs with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs, heparan sulfate (HS and chondroitin sulfate (CS, which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. Methods Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. Results We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. Conclusion Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

  17. CD4+ T-cell lines used to evaluate a Mycobacterium avium subsp. paratuberculosis (MAP) peptide vaccine

    DEFF Research Database (Denmark)

    Lybeck, Kari; Sjurseth, Siri K.; Al-Touama, Zainab;

    The aim of the study was to establish a protocol for generation of MAP-specific T-cell lines and to use these lines for evaluation of a peptide vaccine. A protocol for culturing T-cell lines from peripheral blood of goats naturally infected with MAP was established. CD4+ T cells were positively...... selected using an anti CD4 mAb and Dynabeads. Sorted CD4+ cells were cultivated with purified protein derivative from MAP (PPDj) or E. coli sonicate, IL-2, and IL-15. After two cultivation cycles, T cells were tested for recall responses in a proliferative T-cell assay. T-cell line responses were in...... antigens. T-cell lines were now generated by cultivating CD4+ cells with peptides instead of PPDj. Initially, both healthy and MAP-infected goats were vaccinated with 119 peptides defined by in silico analysis. Cellular responses to the peptides were not detected using standard IFN- γ plasma ELISA. However...

  18. Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell-nucleus targeting.

    Science.gov (United States)

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y Eugene; Sun, Shouheng

    2008-03-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications. PMID:18080259

  19. Stereochemistry Balances Cell Permeability and Solubility in the Naturally Derived Phepropeptin Cyclic Peptides.

    Science.gov (United States)

    Schwochert, Joshua; Lao, Yongtong; Pye, Cameron R; Naylor, Matthew R; Desai, Prashant V; Gonzalez Valcarcel, Isabel C; Barrett, Jaclyn A; Sawada, Geri; Blanco, Maria-Jesus; Lokey, R Scott

    2016-08-11

    Cyclic peptide (CP) natural products provide useful model systems for mapping "beyond-Rule-of-5" (bRo5) space. We identified the phepropeptins as natural product CPs with potential cell permeability. Synthesis of the phepropeptins and epimeric analogues revealed much more rapid cellular permeability for the natural stereochemical pattern. Despite being more cell permeable, the natural compounds exhibited similar aqueous solubility as the corresponding epimers, a phenomenon explained by solvent-dependent conformational flexibility among the natural compounds. When analyzing the polarity of the solution structures we found that neither the number of hydrogen bonds nor the total polar surface area accurately represents the solvation energies of the high and low dielectric conformations. This work adds to a growing number of natural CPs whose solvent-dependent conformational behavior allows for a balance between aqueous solubility and cell permeability, highlighting structural flexibility as an important consideration in the design of molecules in bRo5 chemical space. PMID:27563399

  20. Cytocompatibility of Self-assembled Hydrogel from IKVAV-containing Peptide Amphiphile with Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    SONG Yulin; ZHENG Qixin; GUO Xiaodong; ZHENG Jianfeng

    2009-01-01

    Neural Stem Cells(NSCs)were incubated with self-assembled hydrogel from IKVAV-containing peptide amphiphile(IKVAV-PA)for one week.The cytocompatibility of hydrogel was evaluated.NSCs were seeded in three-dimensional(3D)hydrogels(Experimental Group,EG)or surface of coverslips(Control Group,CG),double-labeled with Calcein-AM and PI.A growth curve of cells was obtained according to CCK-8.TEM study of hydrogel revealed a network of nanofibers. NSCs began to proliferate after 24 h of incubation,and formed bigger neurospheres at 48 h in EG than in CG.Cell proliferation activity was higher in EG than in CG(P<0.05).The self-assembled Hydrogel had good cytocompatibility and promoted the proliferation of NSCs.

  1. Frequency patterns of T-cell exposed motifs in immunoglobulin heavy chain peptides presented by MHCs

    Directory of Open Access Journals (Sweden)

    Robert D. Bremel

    2014-10-01

    Full Text Available Immunoglobulins are highly diverse protein sequences that are processed and presented to T-cells by B-cells and other antigen presenting cells. We examined a large dataset of immunoglobulin heavy chain variable regions (IGHV to assess the diversity of T-cell exposed motifs (TCEM. TCEM comprise those amino acids in a MHC-bound peptide which face outwards, surrounded by the MHC histotope, and which engage the T-cell receptor. Within IGHV there is a distinct pattern of predicted MHC class II binding and a very high frequency of re-use of the TCEMs. The re-use frequency indicates that only a limited number of different cognate T-cells are required to engage many different clonal B-cells. The amino acids in each outward-facing TCEM are intercalated with the amino acids of inward-facing MHC groove-exposed motifs (GEM. Different GEM may have differing, allele-specific, MHC binding affinities. The intercalation of TCEM and GEM in a peptide allows for a vast combinatorial repertoire of epitopes, each eliciting a different response. Outcome of T-cell receptor binding is determined by overall signal strength, which is a function of the number of responding T-cells and the duration of engagement. Hence, the frequency of T-cell exposed motif re-use appears to be an important determinant of whether a T-cell response is stimulatory or suppressive. The frequency distribution of TCEMs implies that somatic hypermutation is followed by clonal expansion that develop along repeated pathways. The observations of TCEM and GEM derived from immunoglobulins suggest a relatively simple, yet powerful, mechanism to correlate T-cell polyspecificity, through re-use of TCEMs, with a very high degree of specificity achieved by combination with a diversity of GEMs. The frequency profile of TCEMs also points to an economical mechanism for maintaining T-cell memory, recall, and self-discrimination based on an endogenously generated profile of motifs.

  2. Peptide nucleic acids arrest the growth of gastric cancer cells SGC7901

    Institute of Scientific and Technical Information of China (English)

    王宽; 张岂凡; 王锡山; 薛英威; 庞达; 傅松滨

    2004-01-01

    Background Peptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells.Methods Heteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay.Results PNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99.4% after 7 days; the rate of cloning inhibition was 98.2% after 8 days;whereas for oligonucleotide groups, at the same concentration, the percentages were 50. 1% and 67. 5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P<0.05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P<0.05). At the same time, the impact on cell morphology was observed.Conclusions The results showed that PNAs are potent antisense reagents. The telomeraseassociated therapies are very promising for the treatment of malignant tumours.

  3. Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration.

    Science.gov (United States)

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

    2016-01-01

    The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. PMID:27509895

  4. Bioconjugated Gold Nanoparticles Penetrate Into Spermatozoa Depending on Plasma Membrane Status.

    Science.gov (United States)

    Barchanski, Annette; Taylor, Ulrike; Sajti, Csaba L; Gamrad, Lisa; Kues, Wilfried A; Rath, Detlef; Barcikowski, Stephan

    2015-09-01

    Spermatozoa are not only essential for animal reproduction they also represent important tools for the manipulation of animal genetics. For instance, the genetic labeling and analysis of spermatozoa could provide a prospective complementation of pre-fertilization diagnosis and could help to prevent the inheritance of defective alleles during artificial insemination or to select beneficial traits in livestock. Spermatozoa feature extremely specialized membrane organization and restricted transport mechanisms making the labeling of genetically interesting DNA-sequences, e.g., with gold nanoparticles, a particular challenge. Here, we present a systematic study on the size-related internalization of ligand-free, monovalent and bivalent polydisperse gold nanoparticles, depending on spermatozoa membrane status. While monovalent conjugates were coupled solely to either negatively-charged oligonucleotides or positively-charged cell-penetrating peptides, bivalent conjugates were functionalized with both molecules simultaneously. The results clearly indicate that the cell membrane of acrosome-intact, bovine spermatozoa was neither permeable to ligand-free or oligonucleotide-conjugated nanoparticles, nor responsive to the mechanisms of cell-penetrating peptides. Interestingly, after acrosome reaction, which comprises major changes in sperm membrane composition, fluidity and charge, high numbers of monovalent and bivalent nanoparticles were found in the postequatorial segment, depicting a close and complex correlation between particle internalization and membrane organization. Additionally, depending on the applied peptide and for nanoparticle sizes < 10 nm even a successive nuclear penetration was observed, making the bivalent conjugates promising for future genetic delivery and sorting issues. PMID:26485929

  5. Full protection of swine against foot-and-mouth disease by a bivalent B-cell epitope dendrimer peptide

    NARCIS (Netherlands)

    Blanco, Esther; Guerra, Beatriz; Torre, de la Beatriz; Defaus, Sira; Dekker, A.; Andreu, D.; Sobrino, Francisco

    2016-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have reported (Cubillos et al., 2008) that a synthetic dendrimeric peptide consisting of four copies of a B-cell epitope [VP1(136–154)] linked through thioether bonds to a T-cell epitope [3A(21–35)] o

  6. Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis

    NARCIS (Netherlands)

    Zhang, R.; Yang, P.Z.; Wu, C.Y.; Jin, H.L.; Li, B.; Huang, X.; Zhou, H.; Gao, Y.; Zhu, L.; Kijlstra, A.

    2006-01-01

    T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a sy

  7. Adjuvanted HLA-supertype restricted subdominant peptides induce new T-cell immunity during untreated HIV-1-infection

    DEFF Research Database (Denmark)

    Karlsson, Ingrid; Brandt, Lea; Vinner, Lasse;

    2013-01-01

    -cell responses specific for one or more vaccine epitopes were induced in 10/10 vaccinees. The responses were dominated by CD107a and MIP1β expression. There were no significant changes in HIV-1 viral load or CD4 T-cell counts. Our study demonstrates that the peptide/CAF01 vaccine is safe and that it is possible...

  8. Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T-cell activation.

    Science.gov (United States)

    Wälchli, Sébastien; Kumari, Shraddha; Fallang, Lars-Egil; Sand, Kine M K; Yang, Weiwen; Landsverk, Ole J B; Bakke, Oddmund; Olweus, Johanna; Gregers, Tone F

    2014-03-01

    Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

  9. Generation in vivo of peptide-specific cytotoxic T cells and presence of regulatory T cells during vaccination with hTERT (class I and II peptide-pulsed DCs

    Directory of Open Access Journals (Sweden)

    Satthaporn Sukchai

    2009-03-01

    Full Text Available Abstract Background Optimal techniques for DC generation for immunotherapy in cancer are yet to be established. Study aims were to evaluate: (i DC activation/maturation milieu (TNF-α +/- IFN-α and its effects on CD8+ hTERT-specific T cell responses to class I epitopes (p540 or p865, (ii CD8+ hTERT-specific T cell responses elicited by vaccination with class I alone or both class I and II epitope (p766 and p672-pulsed DCs, prepare