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Sample records for acid rgd peptides

  1. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won; Lim, Yun Kyong; Kook, Joong-Ki; Cho, Dong-Lyun; Kim, Byung Hoon

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH2 of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  2. Cell-Adhesive Matrices Composed of RGD Peptide-Displaying M13 Bacteriophage/Poly(lactic-co-glycolic acid) Nanofibers Beneficial to Myoblast Differentiation.

    PubMed

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Linhua; Kim, Min Jeong; Kim, Chuntae; Hong, Suck Won; Oh, Jin Woo; Han, Dong-Wook

    2015-10-01

    Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.

  3. Molecular Imaging of Breast Cancer: Role of RGD Peptides.

    PubMed

    Chakravarty, Rubel; Chakraborty, Sudipta; Dash, Ashutosh

    2015-01-01

    Breast cancer is the leading cause of cancer deaths among women of all ages worldwide. With advances in molecular imaging procedures, it has been possible to detect breast cancer in its early stage, determine the extent of the disease to administer appropriate therapeutic protocol and also monitor the effects of treatment. By accurately characterizing the tumor properties and biological processes involved, molecular imaging can play a crucial role in minimizing the morbidity and mortality associated with breast cancer. The integrin αvβ3 plays an important role in breast cancer angiogenesis and is expressed on tumor endothelial cells as well as on some tumor cells. It is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic acid (RGD) tripeptide sequence and therefore RGD peptides can preferentially bind to integrin αvβ3. In this context, targeting tumor vasculature or tumor cells by RGD-based probes is a promising strategy for molecular imaging of breast cancer. Using RGD-based probes, several preclinical studies have employed different imaging modalities such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), ultrasound and optical imaging for visualization of integrin αvβ3 expression in breast cancer models. Limited clinical trials using (18)F-labeled RGD peptides have also been initiated for non-invasive detection and staging of breast cancer. Herein, we provide a comprehensive overview of the latest advances in molecular imaging of breast cancer using RGD peptide-based probes and discuss the challenges and opportunities for advancement of the field. The reported strategies for molecular imaging of breast cancer using RGD peptide-based probes holds promise for making clinically translatable advances that can positively impact the overall diagnostic and therapeutic processes and result in improved quality of life for breast cancer patients.

  4. Tumor-Penetrating iRGD Peptide Inhibits Metastasis

    PubMed Central

    Sugahara, Kazuki N.; Braun, Gary B.; de Mendoza, Tatiana Hurtado; Kotamraju, Venkata Ramana; French, Randall P.; Lowy, Andrew M.; Teesalu, Tambet; Ruoslahti, Erkki

    2014-01-01

    Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The anti-metastatic effect was mediated by the NRP-binding RXXK peptide motif (CendR motif), and not by the integrin-binding RGD motif. iRGD inhibited migration of tumor cells and caused chemorepulsion in vitro in a CendR- and NRP-1-dependent manner. The peptide induced dramatic collapse of cellular processes and partial cell detachment, resulting in the repellent activity. These effects were prominently displayed when the cells were seeded on fibronectin, suggesting a role of CendR in functional regulation of integrins. The anti-metastatic activity of iRGD may provide a significant additional benefit when this peptide is used for drug delivery to tumors. PMID:25392370

  5. RGD based peptide amphiphiles as drug carriers for cancer targeting

    NASA Astrophysics Data System (ADS)

    Saraf, Poonam S.

    Specific interactions of ligands with receptors is one of the approaches for active targeting of anticancer drugs to cancer cells. Over expression of integrin receptors is a physiological manifestation in several cancers and is associated with cancer progression and metastasis, which makes it an attractive target for cancer chemotherapy. The peptide sequence for this integrin recognition is the Arg-Gly-Asp (RGD). Self-assembly offers a unique way of presenting ligands to target receptors for recognition and binding. This study focuses on development of integrin specific peptide amphiphile self-assemblies as carriers for targeted delivery of paclitaxel to αvbeta 3 integrin overexpressing cancers. Amphiphiles composed of conjugates of different analogs of RGD (linear, cyclic or glycosylated) and aliphatic fatty acid with or without 8-amino-3,6-dioxaoctanoic acid (ADA) as linker were synthesized and characterized. The amphiphiles exhibited Critical Micellar Concentration in the range of 7-30 μM. Transmission electron microscopy images revealed the formation of spherical micelles in the size range of 10-40 nm. Forster Resonance Energy Transfer studies revealed entrapment of hydrophobic dyes within a tight micellar core and provided information regarding the cargo exchange within micelles. The RGD micelles exhibited competitive binding with 55% displacement of a bound fluorescent probe by the cyclic RGD micelles. The internalization of fluorescein isothiocynate (FITC) loaded RGD micelles was significantly higher in A2058 melanoma cells compared to free FITC within 20 minutes of incubation at 37°C. The same micelles showed significantly lower internalization at 4°C and on pretreatment with 0.45M sucrose confirming endocytotic uptake of the RGD micellar carriers. The IC50 of paclitaxel in A2058 melanoma cells was lower when treated within RGD micelles as compared to treatment of free drug. On the other hand, IC50 values increased by 2 to 9 fold for micellar treatment

  6. Biomimetic Hybrid Nanofiber Sheets Composed of RGD Peptide-Decorated PLGA as Cell-Adhesive Substrates.

    PubMed

    Shin, Yong Cheol; Lee, Jong Ho; Kim, Min Jeong; Park, Ji Hoon; Kim, Sung Eun; Kim, Jin Su; Oh, Jin-Woo; Han, Dong-Wook

    2015-05-29

    In biomedical applications, there is a need for tissue engineering scaffolds to promote and control cellular behaviors, including adhesion, proliferation and differentiation. In particular, the initial adhesion of cells has a great influence on those cellular behaviors. In this study, we concentrate on developing cell-adhesive substrates applicable for tissue engineering scaffolds. The hybrid nanofiber sheets were prepared by electrospinning poly(lactic-co-glycolic acid) (PLGA) and M13 phage, which was genetically modified to enhance cell adhesion thru expressing RGD peptides on their surface. The RGD peptide is a specific motif of extracellular matrix (ECM) for integrin receptors of cells. RGD peptide-decorated PLGA (RGD-PLGA) nanofiber sheets were characterized by scanning electron microscopy, immunofluorescence staining, contact angle measurement and differential scanning calorimetry. In addition, the initial adhesion and proliferation of four different types of mammalian cells were determined in order to evaluate the potential of RGD-PLGA nanofiber sheets as cell-adhesive substrates. Our results showed that the hybrid nanofiber sheets have a three-dimensional porous structure comparable to the native ECM. Furthermore, the initial adhesion and proliferation of cells were significantly enhanced on RGD-PLGA sheets. These results suggest that biomimetic RGD-PLGA nanofiber sheets can be promising cell-adhesive substrates for application as tissue engineering scaffolds.

  7. Improved tumor-targeting MRI contrast agents: Gd(DOTA) conjugates of a cycloalkane-based RGD peptide

    SciTech Connect

    Park, Ji-Ae; Lee, Yong Jin; Ko, In Ok; Kim, Tae-Jeong; Chang, Yongmin; Lim, Sang Moo; Kim, Kyeong Min; Kim, Jung Young

    2014-12-12

    Highlights: • Development of improved tumor-targeting MRI contrast agents. • To increase the targeting ability of RGD, we developed cycloalkane-based RGD peptides. • Gd(DOTA) conjugates of cycloalkane-based RGD peptide show improved tumor signal enhancement in vivo MR images. - Abstract: Two new MRI contrast agents, Gd-DOTA-c(RGD-ACP-K) (1) and Gd-DOTA-c(RGD-ACH-K) (2), which were designed by incorporating aminocyclopentane (ACP)- or aminocyclohexane (ACH)-carboxylic acid into Gd-DOTA (gadolinium-tetraazacyclo dodecanetetraacetic acid) and cyclic RGDK peptides, were synthesized and evaluated for tumor-targeting ability in vitro and in vivo. Binding affinity studies showed that both 1 and 2 exhibited higher affinity for integrin receptors than cyclic RGDyK peptides, which were used as a reference. These complexes showed high relaxivity and good stability in human serum and have the potential to improve target-specific signal enhancement in vivo MR images.

  8. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins

    PubMed Central

    1995-01-01

    Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*- peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD. PMID:7657703

  9. New Methods for Labeling RGD Peptides with Bromine-76

    PubMed Central

    Lang, Lixin; Li, Weihua; Jia, Hong-Mei; Fang, De-Cai; Zhang, Shushu; Sun, Xilin; Zhu, Lei; Ma, Ying; Shen, Baozhong; Kiesewetter, Dale O.; Niu, Gang; Chen, Xiaoyuan

    2011-01-01

    Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[76Br]bromo-2,6-dimethoxybenzoate ([76Br]SBDMB) was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB) moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [76Br]-BrE[c(RGDyK)]2 and [76Br]-BrDMB-E[c(RGDyK)]2. An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK) and the brominating agent CH3COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK) and E[c(RGDyK)]2 using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [76Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK) and c(RGDyK) were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [76Br]Br-E[c(RGDyK)]2 and [76Br]BrDMB-E[c(RGDyK)]2 showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers was

  10. Graphene oxide-stimulated myogenic differentiation of C2C12 cells on PLGA/RGD peptide nanofiber matrices

    NASA Astrophysics Data System (ADS)

    Shin, Y. C.; Lee, J. H.; Kim, M. J.; Hong, S. W.; Oh, J.-W.; Kim, C.-S.; Kim, B.; Hyun, J. K.; Kim, Y.-J.; Han, D.-W.

    2015-07-01

    During the last decade, much attention has been paid to graphene-based nanomaterials because they are considered as potential candidates for biomedical applications such as scaffolds for tissue engineering and substrates for the differentiation of stem cells. Until now, electrospun matrices composed of various biodegradable copolymers have been extensively developed for tissue engineering and regeneration; however, their use in combination with graphene oxide (GO) is novel and challenging. In this study, nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 phage with RGD peptide displayed on its surface (RGD peptide-M13 phage) were prepared as extracellular matrix (ECM)-mimicking substrates. RGD peptide is a tripeptide (Arg-Gly-Asp) found on ECM proteins that promotes various cellular behaviors. The physicochemical properties of PLGA and RGD peptide-M13 phage (PLGA/RGD peptide) nanofiber matrices were characterized by atomic force microscopy, Fourier-transform infrared spectroscopy and thermogravimetric analysis. In addition, the growth of C2C12 mouse myoblasts on the PLGA/RGD peptide matrices was examined by measuring the metabolic activity. Moreover, the differentiation of C2C12 mouse myoblasts on the matrices when treated with GO was evaluated. The cellular behaviors, including growth and differentiation of C2C12 mouse myoblasts, were substantially enhanced on the PLGA/RGD peptide nanofiber matrices when treated with GO. Overall, these findings suggest that the PLGA/RGD peptide nanofiber matrices can be used in combination with GO as a novel strategy for skeletal tissue regeneration.

  11. Highly Water-soluble, Near-infrared Emissive BODIPY Polymeric Dye Bearing RGD Peptide Residues for Cancer Imaging

    PubMed Central

    Zhu, Shilei; Zhang, Jingtuo; Janjanam, Jagadeesh; Bi, Jianheng; Vegesna, Giri; Tiwari, Ashutosh; Luo, Fen-Tair; Wei, Jianjun

    2012-01-01

    Near-infrared emissive BODIPY polymeric dye bearing cancer-homing cyclic arginine-glycine-aspartic acid (RGD) peptide residues (polymer B) was prepared by post-polymerization functionalization of BODIPY polymeric dye bearing bromo groups through tetra(ethylene glycol tethered spacers (polymer A) with thiol-functionalized RGD cancer-homing peptide through thioether bonds under a mild basic condition. Polymer B possesses excellent water solubility, good photostability, biocompatibility and resistance to nonspecific interactions to normal endothelial cells, and can efficiently detect breast tumor cells through specific cooperative binding of cancer-homing RGD peptides to αVβ3 integrins of cancer cells while its parent polymer Awith outRGD residues fails to target cancer cells. PMID:23245906

  12. Topical ocular delivery to laser-induced choroidal neovascularization by dual internalizing RGD and TAT peptide-modified nanoparticles

    PubMed Central

    Chu, Yongchao; Chen, Ning; Yu, Huajun; Mu, Hongjie; He, Bin; Hua, Hongchen; Wang, Aiping; Sun, Kaoxiang

    2017-01-01

    A nanoparticle (NP) was developed to target choroidal neovascularization (CNV) via topical ocular administration. The NPs were prepared through conjugation of internalizing arginine-glycine-aspartic acid RGD (iRGD; Ac-CCRGDKGPDC) and transactivated transcription (TAT) (RKKRRQRRRC) peptide to polymerized ethylene glycol and lactic-co-glycolic acid. The iRGD sequence can specifically bind with integrin αvβ3, while TAT facilitates penetration through the ocular barrier. 1H nuclear magnetic resonance and high-performance liquid chromatography demonstrated that up to 80% of iRGD and TAT were conjugated to poly(ethylene glycol)– poly(lactic-co-glycolic acid). The resulting particle size was 67.0±1.7 nm, and the zeta potential of the particles was −6.63±0.43 mV. The corneal permeation of iRGD and TAT NPs increased by 5.50- and 4.56-fold compared to that of bare and iRGD-modified NPs, respectively. Cellular uptake showed that the red fluorescence intensity of iRGD and TAT NPs was highest among primary NPs and iRGD- or TAT-modified NPs. CNV was fully formed 14 days after photocoagulation in Brown Norway (BN) rats as shown by optical coherence tomography and fundus fluorescein angiography analyses. Choroidal flat mounts in BN rats showed that the red fluorescence intensity of NPs followed the order of iRGD and TAT NPs > TAT-modified NPs > iRGD-modified NPs > primary NPs. iRGD and TAT dual-modified NPs thus displayed significant targeting and penetration ability both in vitro and in vivo, indicating that it is a promising drug delivery system for managing CNV via topical ocular administration. PMID:28260884

  13. RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

    PubMed

    He, Xuedan; Alves, Carla S; Oliveira, Nilsa; Rodrigues, João; Zhu, Jingyi; Bányai, István; Tomás, Helena; Shi, Xiangyang

    2015-01-01

    Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells.

  14. RGD-peptide modified alginate by a chemoenzymatic strategy for tissue engineering applications.

    PubMed

    Sandvig, Ioanna; Karstensen, Kristin; Rokstad, Anne Mari; Aachmann, Finn Lillelund; Formo, Kjetil; Sandvig, Axel; Skjåk-Bræk, Gudmund; Strand, Berit Løkensgard

    2015-03-01

    One of the main challenges in tissue engineering and regenerative medicine is the ability to maintain optimal cell function and survival post-transplantation. Biomaterials such as alginates are commonly used for immunoisolation, while they may also provide structural support to the cell transplants by mimicking the extracellular matrix. In this study, arginine-glycine-aspartate (RGD)-peptide-coupled alginates of tailored composition were produced by adopting a unique chemoenzymatic strategy for substituting the nongelling mannuronic acid on the alginate. Alginates with and without RGD were produced with high and low content of G. Using carbodiimide chemistry 0.1-0.2% of the sugar units were substituted by peptide. Furthermore, the characterization by (1)H-nuclear magnetic resonance (NMR) revealed by-products from the coupling reaction that partly could be removed by coal filtration. Olfactory ensheathing cells (OECs) and myoblasts were grown in two-dimensional (2D) and 3D cultures of RGD-peptide modified or unmodified alginates obtained by the chemoenzymatically strategy and compared to native alginate. Both OECs and myoblasts adhered to the RGD-peptide modified alginates in 2D cultures, forming bipolar protrusions. OEC encapsulation resulted in cell survival for up to 9 days, thus demonstrating the potential for short-term 3D culture. Myoblasts showed long-term survival in 3D cultures, that is, up to 41 days post encapsulation. The RGD modifications did not result in marked changes in cell viability in 3D cultures. We demonstrate herein a unique technique for tailoring peptide substituted alginates with a precise and flexible composition, conserving the gel forming properties relevant for the use of alginate in tissue engineering.

  15. Cyclopropane pipecolic acids as templates for linear and cyclic peptidomimetics: application in the synthesis of an Arg-Gly-Asp (RGD)-containing peptide as an αvβ3 integrin ligand.

    PubMed

    Sernissi, Lorenzo; Petrović, Martina; Scarpi, Dina; Guarna, Antonio; Trabocchi, Andrea; Bianchini, Francesca; Occhiato, Ernesto G

    2014-08-25

    The synthesis and evaluation of substituted cyclopropane pipecolic acids (CPA) as conformationally restricted templates for linear and cyclic peptidomimetics is reported. A variety of differently substituted (poly)hydroxy- and amino-2-azabicyclo[4.1.0]heptane-1-carboxylic acids were prepared by means of the Pd-catalyzed methoxycarbonylation of suitably functionalized lactam-derived enol phosphates, followed by OH-directed cyclopropanation. CPAs were successfully introduced into a linear peptide sequence to assess the cis/trans isomerism about the pipecolic acid peptide bond, and in a cyclic peptidomimetic that bore the Arg-Gly-Asp (RGD) sequence, which displayed nanomolar activity as antagonist of the αvβ3 integrin in M21 human melanoma cells. Thus, CPAs appear to be suitable for the generation of novel peptidomimetics for drug discovery.

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

    PubMed

    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.

  17. Immobilization of RGD peptide on HA coating through a chemical bonding approach.

    PubMed

    Yang, Chunli; Cheng, Kui; Weng, Wenjian; Yang, Chunyu

    2009-11-01

    In this work, Arg-Gly-Asp (RGD) sequence containing peptide was immobilized on hydroxyapatite (HA) coatings through a chemical bonding approach in two steps, surface modification with 3-aminopropyltriethoxysilane (APTES) and RGD immobilization. The results indicate that RGD has been successfully immobilized on HA coatings. Comparing with physical adsorption coatings, the chemically bonded RGD on the coatings shows much better anti-wash-out ability. Since RGD is able to recognize cell-membrane integrins on biointerfaces, the present method will be an effective way to favor interaction of cells with HA coatings.

  18. RGD peptide-displaying M13 bacteriophage/PLGA nanofibers as cell-adhesive matrices for smooth muscle cells

    NASA Astrophysics Data System (ADS)

    Shin, Yong Cheol; Lee, Jong Ho; Jin, Oh Seong; Lee, Eun Ji; Jin, Lin Hua; Kim, Chang-Seok; Hong, Suck Won; Han, Dong-Wook; Kim, Chuntae; Oh, Jin-Woo

    2015-01-01

    Extracellular matrices (ECMs) are network structures that play an essential role in regulating cellular growth and differentiation. In this study, novel nanofibrous matrices were fabricated by electrospinning M13 bacteriophage and poly(lactic- co-glycolic acid) (PLGA) and were shown to be structurally and functionally similar to natural ECMs. A genetically-engineered M13 bacteriophage was constructed to display Arg-Gly-Asp (RGD) peptides on its surface. The physicochemical properties of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage)/PLGA nanofibers were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. We used immunofluorescence staining to confirm that M13 bacteriophages were homogenously distributed in RGD-M13 phage/PLGA matrices. Furthermore, RGD-M13 phage/PLGA nanofibrous matrices, having excellent biocompatibility, can enhance the behaviors of vascular smooth muscle cells. This result suggests that RGD-M13 phage/PLGA nanofibrous matrices have potentials to serve as tissue engineering scaffolds.

  19. DFT computational study of the RGD peptide interaction with the rutile TiO2 (110) surface

    NASA Astrophysics Data System (ADS)

    Muir, J. M. R.; Costa, D.; Idriss, H.

    2014-06-01

    Planewave DFT calculations including ab initio molecular dynamics (AIMD) were used to model the adsorption of a biologically relevant peptide sequence, arginine-glycine-aspartic acid (RGD), upon a rutile TiO2 (110) surface. It was found that binding is solely through the aspartic acid end of the RGD. The carboxy groups bind through dissociative bridging and molecular forms, similar to formic acid. The energy of adsorption is much smaller (0.5-0.77 eV) than seen for formic acid and the molecular adsorption is the strongest adsorption mode. Neutral adsorption is favoured over zwitterionic adsorption and adsorption through the carboxy group of the aspartic acid side chain rather than the terminal carboxy group is favoured due to a configuration allowing an additional surface-carbonyl bond. The RGD backbone is not significantly disrupted upon adsorption.

  20. Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

    PubMed

    Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

    2016-09-10

    A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.

  1. Application of RGD-containing peptides as imaging probes for alphavbeta3 expression.

    PubMed

    Dijkgraaf, Ingrid; Beer, Ambros J; Wester, Hans-Jurgen

    2009-01-01

    Integrin alphavbeta3 plays a pivotale role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glysine-aspartic acid (RGD) tripeptide sequence (e.g. vitronectin, fibronectin). Alphavbeta3 is overexpressed on activated endothelial cells during tumor-induced angiogenesis, whereas it is absent on quiescent endothelial cells and normal tissues. Furthermore, alphavbeta3 is expressed on various tumor cell lines. Due to this restricted expression of alphavbeta3 in tumors, alphavbeta3 is considered a suitable receptor for tumor targeting. In the past decade, several RGD-containing peptide antagonists have been evaluated for monitoring alphavbeta3 expression using SPECT, PET, MRI, OI and US. Molecular imaging tracers for this integrin receptor could be used to noninvasively visualize alphavbeta3 expression in tumors. Noninvasive determination of alphavbeta3 expression potentially can be used to monitor treatment response to antiangiogenic drugs or even to select patients likely to respond to treatment with antiangiogenic drugs. In this review a brief overview on the currently used RGD-containing peptides as imaging probes for noninvasive visualization of alphavbeta3 expression using PET, SPECT, MRI, OI and US is given.

  2. [The synthesis of RGD peptide derivatives containing glutaric and adipic residues].

    PubMed

    Vigorov, A Iu; Demin, A M; Nizova, I A; Krasnov, V P

    2014-01-01

    A method of the synthesis of RGD peptide derivatives containing glutaric or adipic residues linked with α-amino group of L-arginine and allowing carrying out their coupling with other biomolecules and nanoparticles.

  3. Novel Approach to Prepare {sup 99m}Tc-Based Multivalent RGD Peptides

    SciTech Connect

    Shuang Liu

    2012-10-24

    This project presents a novel approach to prepare the {sup 99m}Tc-bridged multivalent RGD (arginine-glycine-aspartate) peptides. This project will focus on fundamentals of {sup 99m}Tc radiochemistry. The main objective of this project is to demonstrate the proof-of-principle for the proposed radiotracers. Once a kit formulation is developed for preparation of the {sup 99m}Tc-bridged multivalent RGD peptides, various tumor-bearing animal models will be used to evaluate their potential for SPECT (single photon-emission computed tomography) imaging of cancer. We have demonstrated that (1) multimerization of cyclic RGD peptides enhances the integrin {alpha}{sub v}{beta}{sub 3} bonding affinity and radiotracer tumor uptake; (2) addition of G{sub 3} or PEG{sub 4} linkers makes it possible for two RGD motifs in 3P-RGD{sub 2} and 3G-RGD{sub 2} to achieve simultaneous integrin {alpha}{sub v}{beta}{sub 3} binding; and (3) multimers are actually bivalent (not multivalent), the presence of extra RGD motifs can enhance the tumor retention time of the radiotracer.

  4. Chitosan microsphere scaffold tethered with RGD-conjugated poly(methacrylic acid) brushes as effective carriers for the endothelial cells.

    PubMed

    Yang, Zhenyi; Yuan, Shaojun; Liang, Bin; Liu, Yang; Choong, Cleo; Pehkonen, Simo O

    2014-09-01

    Endothelial cell-matrix interactions play a vital role in promoting vascularization of engineered tissues. The current study reports a facile and controllable method to develop a RGD peptide-functionalized chitosan microsphere scaffolds for rapid cell expansion of human umbilical vein endothelial cells (HUVECs). Functional poly(methacrylic acid) (PMAA) brushes are grafted from the chitosan microsphere surfaces via surface-initiated ATRP. Subsequent conjugation of RGD peptides on the pendent carboxyl groups of PMAA side chain is accomplished by carbodiimide chemistry to facilitate biocompatibility of the 3D CS scaffolding system. In vitro cell-loading assay of HUVECs exhibits a significant improvment of cell adhesion, spreading, and proliferation on the RGD peptide-immobilized CS microsphere surfaces.

  5. Systemic Administration of siRNA via cRGD-containing Peptide

    PubMed Central

    Huang, Yuanyu; Wang, Xiaoxia; Huang, Weiyan; Cheng, Qiang; Zheng, Shuquan; Guo, Shutao; Cao, Huiqing; Liang, Xing-Jie; Du, Quan; Liang, Zicai

    2015-01-01

    Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment. PMID:26300278

  6. Dual targeting strategy of magnetic nanoparticle-loaded and RGD peptide-activated stimuli-sensitive polymeric micelles for delivery of paclitaxel

    NASA Astrophysics Data System (ADS)

    Lin, Meng Meng; Kang, Yoon Joong; Sohn, Youngjoo; Kim, Do Kyung

    2015-06-01

    A double targeting strategy of anti-neoplastic agent paclitaxel (PTX) was developed by incorporating magnetic nanoparticles and RGD peptide for enhanced cell cytotoxicity effect at lower dosage. A dual targeting mechanism including magnetic targeting and RGD ligand-specific targeting enhanced the overall cytotoxicity and reduced the effective dosage of PTX to achieve enhanced and sustained release of PTX in vitro. We addressed the issues of water-insolubility of oleic acid (OA)-stabilized SPIONs and low incorporation efficiency of hydrophobic PTX with SPION nanocarriers by using an amphiphilic polymer poly[( N-isopropylacrylamide-r-acrylamide)-b- l-lactic acid] (PNAL) as micelle-forming materials. A targeting moiety, GGGGRGD peptide, a RGD sequence-containing peptide with a short linker, is attached to the surface of PNAL-SPIONs via a homo-crosslinker. Confocal microscopy image analysis revealed that the cellular uptake was increased from (1.5 ± 0.5 % (PNAL) to 11.7 ± 0.8 % (RGD-PNAL-SPIONs) at 6 h incubation, once both RGD peptide and magnetic force attraction were incorporated into the carriers. Such multi-targeting nanocarriers showed promising potential in cancer-oriented diagnosis and therapy.

  7. Toward the development of a novel non-RGD cyclic peptide drug conjugate for treatment of human metastatic melanoma

    PubMed Central

    Redko, Boris; Tuchinsky, Helena; Segal, Tamar; Tobi, Dror; Luboshits, Galia; Ashur-Fabian, Osnat; Pinhasov, Albert; Gerlitz, Gabi; Gellerman, Gary

    2017-01-01

    The newly discovered short (9 amino acid) non-RGD S-S bridged cyclic peptide ALOS-4 (H-cycl(Cys-Ser-Ser-Ala-Gly-Ser-Leu-Phe-Cys)-OH), which binds to integrin αvβ3 is investigated as peptide carrier for targeted drug delivery against human metastatic melanoma. ALOS4 binds specifically the αvβ3 overexpressing human metastatic melanoma WM-266-4 cell line both in vitro and in ex vivo assays. Coupling ALOS4 to the topoisomerase I inhibitor Camptothecin (ALOS4-CPT) increases the cytotoxicity of CPT against human metastatic melanoma cells while reduces dramatically the cytotoxicity against non-cancerous cells as measured by the levels of γH2A.X, active caspase 3 and cell viability. Moreover, conjugating ALOS4 to CPT even increases the chemo-stability of CPT under physiological pH. Bioinformatic analysis using Rosetta platform revealed potential docking sites of ALOS4 on the αvβ3 integrin which are distinct from the RGD binding sites. We propose to use this specific non-RGD cyclic peptide as the therapeutic carrier for conjugation of drugs in order to improve efficacy and reduce toxicity of currently available treatments of human malignant melanoma. PMID:27768593

  8. MicroPET Imaging of Integrin αvβ3 Expressing Tumors Using 89Zr-RGD Peptides

    PubMed Central

    Jacobson, Orit; Zhu, Lei; Niu, Gang; Weiss, Ido D.; Szajek, Lawrence P.; Ma, Ying; Sun, Xilin; Yan, Yongjun; Kiesewetter, Dale O.; Liu, Shuang; Chen, Xiaoyuan

    2011-01-01

    Purpose The dimeric transmembrane integrin, αvβ3, is a well-investigated target by different imaging modalities through suitably labeled arginine–glycine–aspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG3-c(RGDfK) (denoted as FK-PEG3), E[c(RGDfK)]2 (denoted as [FK]2), and PEG4-E[PEG4-c(RGDfK)]2 (denoted as [FK]2-3PEG4), with 89Zr (t1/2=78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin αvβ3. Methods The Df conjugated RGD peptides were subjected to integrin αvβ3 binding assay in vitro using MDA-MB-435 breast cancer cells. The 89Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model. Results All four tracers, 89Zr-Df-FK, 89Zr-Df-FK-PEG3, 89Zr-Df-[FK]2, and 89Zr-Df-[FK]2-3PEG4, were labeled in high radiochemical yield (89±4%) and high specific activity (4.07–6 MBq/µg). Competitive binding assay with 125I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin αvβ3 binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of 89Zr-Df-[FK]2 and 89Zr-Df-[FK]2-3PEG4 was significantly higher (4.32±1.73%ID/g and 4.72±0.66%ID/g, respectively, at 2 h post-injection) than the monomers 89Zr-Df-FK and 89Zr-Df-FK-PEG3 (1.97±0.38%ID/g and 1.57±0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, 89Zr-Df-[FK]2-3PEG4 gave the highest tumor-to-background ratio (18.21±2.52 at 2 h post-injection and 19.69±3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the

  9. Cyclic RGD peptide incorporation on phage major coat proteins for improved internalization by HeLa cells.

    PubMed

    Choi, Dong Shin; Jin, Hyo-Eon; Yoo, So Young; Lee, Seung-Wuk

    2014-02-19

    Delivering therapeutic materials or imaging reagents into specific tumor tissues is critically important for development of novel cancer therapeutics and diagnostics. Genetically engineered phages possess promising structural features to develop cancer therapeutic materials. For cancer targeting purposes, we developed a novel engineered phage that expressed cyclic RGD (cRGD) peptides on the pVIII major coat protein using recombinant DNA technology. Using a type 88 phage engineering approach, which inserts a new gene to express additional major coat protein in the noncoding region of the phage genome, we incorporated an additional pVIII major coat protein with relatively bulky cRGD and assembled heterogeneous major coat proteins on the F88.4 phage surfaces. With IPTG control, we could tune different numbers of cRGD peptide displayed on the phage particles up to 140 copies. The resulting phage with cRGD on the recombinant pVIII protein exhibited enhanced internalization efficiency into HeLa cells in a ligand density and conformational structure dependent manner when comparing with the M13 phages modified with either linear RGD on pVIII or cRGD on pIII. Our cRGD peptide engineered phage could be useful for cancer therapy or diagnostic purposes after further modifying the phage with drug molecules or contrast reagents in the future.

  10. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond.

    PubMed

    Hersel, Ulrich; Dahmen, Claudia; Kessler, Horst

    2003-11-01

    Since RGD peptides (R: arginine; G: glycine; D: aspartic acid) have been found to promote cell adhesion in 1984 (Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule, Nature 309 (1984) 30), numerous materials have been RGD functionalized for academic studies or medical applications. This review gives an overview of RGD modified polymers, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on polymers. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed.

  11. Synthesis and characterization of acrylic terpolymers with RGD peptides for biomedical applications.

    PubMed

    Fussell, Garland W; Cooper, Stuart L

    2004-07-01

    The goal of this research was to design a biomaterial, using acrylic terpolymers, which could support endothelial cells and function in small diameter vascular graft applications. Hexyl methacrylate (HMA) and octyl methacrylate (OMA) were used as comonomers to produce a material with a low glass transition temperature (T(g)). Methacrylic acid (MAA) was used to provide ionic character, and methyl methacrylate (MMA) was selected because of its wide usage in biomedical applications. Cation neutralization was employed to modify the mechanical properties. RGD-based peptide sequences were attached to promote endothelial cell adhesion, because vascular grafts seeded with endothelial cells have fewer problems with thrombosis. The two methods used to incorporate peptide sequences were a chain transfer reaction during polymerization, and a coupling reaction attaching the peptides to carboxyl groups on the polymer after polymerization. The compositions that produced T(g)s of approximately 0 degrees C were 75 mol% OMA and 92 mol% HMA. The Young's modulus of the HMA copolymer was approximately 0.37 MPa, well below the desired value of 0.9 MPa. Likewise, the Young's modulus of approximately 0.50 MPa for the OMA copolymer was also below the desired value. After partial neutralization with sodium cations, the Young's moduli increased to approximately 0.93 and 0.99 MPa, respectively. The chain transfer reaction lowered the molecular weights and mechanical properties of the copolymers, while the coupling reaction method had little effect on these properties. The chain transfer method appears to be a promising one-step method to produce polymers with a wide range of peptide concentrations.

  12. Intestinal anti-inflammatory effects of RGD-functionalized silk fibroin nanoparticles in trinitrobenzenesulfonic acid-induced experimental colitis in rats

    PubMed Central

    Rodriguez-Nogales, Alba; Algieri, Francesca; De Matteis, Laura; Lozano-Perez, A. Abel; Garrido-Mesa, Jose; Vezza, Teresa; de la Fuente, J M.; Cenis, Jose Luis; Gálvez, Julio; Rodriguez-Cabezas, Maria Elena

    2016-01-01

    Background Current treatment of inflammatory bowel disease is based on the use of immunosuppressants or anti-inflammatory drugs, which are characterized by important side effects that can limit their use. Previous research has been performed by administering these drugs as nanoparticles that target the ulcerated intestinal regions and increase their bioavailability. It has been reported that silk fibroin can act as a drug carrier and shows anti-inflammatory properties. Purpose This study was designed to enhance the interaction of the silk fibroin nanoparticles (SFNs) with the injured intestinal tissue by functionalizing them with the peptide motif RGD (arginine–glycine–aspartic acid) and to evaluate the intestinal anti-inflammatory properties of these RGD-functionalized silk fibroin nanoparticles (RGD-SFNs) in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis. Materials and methods SFNs were prepared by nanoprecipitation in methanol, and the linear RGD peptide was linked to SFNs using glutaraldehyde as the crosslinker. The SFNs (1 mg/rat) and RGD-SFNs (1 mg/rat) were administered intrarectally to TNBS-induced colitic rats for 7 days. Results The SFN treatments ameliorated the colonic damage, reduced neutrophil infiltration, and improved the compromised oxidative status of the colon. However, only the rats treated with RGD-SFNs showed a significant reduction in the expression of different pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and IL-12) and inducible nitric oxide synthase in comparison with the TNBS control group. Moreover, the expression of both cytokine-induced neutrophil chemoattractant-1 and monocyte chemotactic protein-1 was significantly diminished by the RGD-SFN treatment. However, both treatments improved the intestinal wall integrity by increasing the gene expression of some of its markers (trefoil factor-3 and mucins). Conclusion SFNs displayed intestinal anti-inflammatory properties in the TNBS model of colitis in rats

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

    PubMed Central

    Zuo, Hou Dong; Yao, Wei Wu; Chen, Tian Wu; Zhu, Jiang; Zhang, Juan Juan; Pu, Yu; Liu, Gang; Zhang, Xiao Ming

    2014-01-01

    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. PMID:24977163

  14. The effect of superparamagnetic iron oxide with iRGD peptide on the labeling of pancreatic cancer cells in vitro: a preliminary study.

    PubMed

    Zuo, Hou Dong; Yao, Wei Wu; Chen, Tian Wu; Zhu, Jiang; Zhang, Juan Juan; Pu, Yu; Liu, Gang; Zhang, Xiao Ming

    2014-01-01

    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.

  15. Conjugation with RGD Peptides and Incorporation of Vascular Endothelial Growth Factor Are Equally Efficient for Biofunctionalization of Tissue-Engineered Vascular Grafts

    PubMed Central

    Antonova, Larisa V.; Seifalian, Alexander M.; Kutikhin, Anton G.; Sevostyanova, Victoria V.; Matveeva, Vera G.; Velikanova, Elena A.; Mironov, Andrey V.; Shabaev, Amin R.; Glushkova, Tatiana V.; Senokosova, Evgeniya A.; Vasyukov, Georgiy Yu.; Krivkina, Evgeniya O.; Burago, Andrey Yu.; Kudryavtseva, Yuliya A.; Barbarash, Olga L.; Barbarash, Leonid S.

    2016-01-01

    The blend of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) has recently been considered promising for vascular tissue engineering. However, it was shown that PHBV/PCL grafts require biofunctionalization to achieve high primary patency rate. Here we compared immobilization of arginine–glycine–aspartic acid (RGD)-containing peptides and the incorporation of vascular endothelial growth factor (VEGF) as two widely established biofunctionalization approaches. Electrospun PHBV/PCL small-diameter grafts with either RGD peptides or VEGF, as well as unmodified grafts were implanted into rat abdominal aortas for 1, 3, 6, and 12 months following histological and immunofluorescence assessment. We detected CD31+/CD34+/vWF+ cells 1 and 3 months postimplantation at the luminal surface of PHBV/PCL/RGD and PHBV/PCL/VEGF, but not in unmodified grafts, with the further observation of CD31+CD34−vWF+ phenotype. These cells were considered as endothelial and produced a collagen-positive layer resembling a basement membrane. Detection of CD31+/CD34+ cells at the early stages with subsequent loss of CD34 indicated cell adhesion from the bloodstream. Therefore, either conjugation with RGD peptides or the incorporation of VEGF promoted the formation of a functional endothelial cell layer. Furthermore, both modifications increased primary patency rate three-fold. In conclusion, both of these biofunctionalization approaches can be considered as equally efficient for the modification of tissue-engineered vascular grafts. PMID:27854352

  16. Indirect coating of RGD peptides using a poly-L-lysine spacer enhances jaw periosteal cell adhesion, proliferation, and differentiation into osteogenic tissue.

    PubMed

    Ardjomandi, N; Klein, C; Kohler, K; Maurer, A; Kalbacher, H; Niederländer, J; Reinert, S; Alexander, D

    2012-08-01

    The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs.

  17. Development of a novel cyclic RGD peptide for multiple targeting approaches of liposomes to tumor region.

    PubMed

    Amin, Mohamadreza; Mansourian, Mercedeh; Koning, Gerben A; Badiee, Ali; Jaafari, Mahmoud Reza; ten Hagen, Timo L M

    2015-12-28

    Liposomes containing cytotoxic agents and targeted with Arg-Gly-Asp based peptides have frequently been used against αvβ3 integrin on tumor neovasculature. However, like many other ligand modified liposomes these preparations suffered from enhanced uptake by the reticulo endothelial system (RES) and off-targeted interaction with integrin receptors vastly expressed in normal organs causing poor biodistribution and toxic effects. Here we mainly focus on development of a RGD-modified liposomal delivery system to enhance both targeting selectivity and tumor uptake. First, sterically stabilized liposomal doxorubicin (SSLD) prepared and decorated with cRGDfK and RGDyC peptides differ in their physical properties. Stability assessments as well as in vitro and in vivo studies revealed that increasing the peptide hydrophobicity promotes the therapeutic efficacy of RGD-SSLD in a C-26 tumor model due to decreased recognition by RES and opsonization and limited off-targeted interactions. Then a novel N-methylated RGD peptide was designed and its capability in targeting integrin presenting cells was comprehensively assessed both in vitro and in vivo. RGDf[N-methyl]C promotes the liposome internalization by HUVEC via integrin mediated endocytosis. Intravital microscopy in window chamber bearing mice illustrated the capability of RGDf[N-methyl]C-liposomes in targeting both tumor vasculature and tumor cells in murine B16F0 and human BLM tumor models. Quantitative biodistribution in mice bearing B16F0 tumor revealed its high affinity to tumor with no considerable affinity to normal organs. Treatment by high dose of RGDf[N-methyl]C-SSLD was found more effective than non-targeted SSLD and no toxic side effect was observed. In conclusion, the RGDf[N-methyl]C-liposome was found promising in targeting tumor vasculature as well as other cells inside the tumor.

  18. microPET Imaging of Glioma Integrin (alpha-v, beta-3) Expression Using Cu-64-Labeled Tetrameric RGD Peptide

    SciTech Connect

    Wu, Yun; Zhang, , Xianzhong; Xiong, , Zhengming; Cheng, Zhen; Fisher, Darrell R.; Liu, Shu-hong; Gambhir, Sanjiv S.; Chen, Xiaoyuan

    2005-10-01

    Integrins ?v?3 and ?v?5 play a critical role in tumor-induced angiogenesis and metastasis, and have become promising diagnostic indicators and therapeutic targets of tumors. Radiolabeled RGD peptides that are integrin-specific may be used for non-invasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy. We previously conjugated a series of mono- and dimeric RGD peptides with 1,4,7,10-tetraazacyclododecane-N, N?,N??,N???-tetraacetic acid (DOTA) and labeled these with copper-64 for microPET imaging in various mouse xenograft models. The copper-64 tracers showed ?v?3-selective tumor uptake, but the magnitude of tumor uptake was relatively low, the tumor washout was rapid, and non-target organ/tissue retention was high. In this study we developed a tetrameric RGD peptide tracer 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 for positron emission tomography (PET) imaging of integrin ?v?3 expression in a subcutaneous U87MG glioma xenograft model in female athymic nude mice. The RGD tetramer showed significantly higher integrin binding affinity than the corresponding mono- and dimeric RGD analogs, most likely due to polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 ? 0.01%ID/g at 30 min and 0.21 ? 0.01 %ID/g at 4 h postinjection (p.i.), respectively) and predominantly renal excretion. Tumor uptake was rapid and high and the tumor washout was slow (9.93 ? 1.05 %ID/g at 30 min p.i. and 4.56 ? 0.51 %ID/g at 24 h post-injection). The metabolic stability of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70, 58, 51 and 26 percent, respectively. Non-invasive microPET imaging studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results

  19. The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells.

    PubMed

    Yang, Fan; Williams, Christopher G; Wang, Dong-An; Lee, Hyukjin; Manson, Paul N; Elisseeff, Jennifer

    2005-10-01

    Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.

  20. iRGD tumor-penetrating peptide-modified oncolytic adenovirus shows enhanced tumor transduction, intratumoral dissemination and antitumor efficacy.

    PubMed

    Puig-Saus, C; Rojas, L A; Laborda, E; Figueras, A; Alba, R; Fillat, C; Alemany, R

    2014-08-01

    Endovenously administered oncolytic viruses extravasate and penetrate poorly into tumors. iRGD is a cyclic peptide that enhances tumor penetration when conjugated or coadministered with different types of molecules such as drugs, nanoparticles or phages. iRGD-mediated tumor penetration occurs in three steps: binding to αv-integrins on tumor vasculature or tumor cells, exposure by proteolysis of a C-terminal motif that binds to neuropilin-1 (NRP-1) and cell internalization. We have genetically inserted the iRGD peptide in the fiber C terminus of ICOVIR15K, an oncolytic tumor-retargeted adenovirus to increase its tumor penetration. In vitro, NRP-1 interaction improved binding and internalization of the virus in different cancer cells overexpressing integrins and NRP-1. However, such NRP-1-mediated internalization did not affect transduction or cytotoxicity. In vivo, iRGD did not change the normal organ transduction pattern, with liver and spleen as main targeted organs. In tumors, however, iRGD enhanced transduction and early adenovirus dissemination through the tumor mass leading to an improved antitumor efficacy.

  1. RGD peptides and monoclonal antibodies, antagonists of alpha(v)-integrin, enter the cells by independent endocytic pathways.

    PubMed

    Castel, S; Pagan, R; Mitjans, F; Piulats, J; Goodman, S; Jonczyk, A; Huber, F; Vilaró, S; Reina, M

    2001-12-01

    Cyclic synthetic peptides containing the arginine-glycine-aspartate motif (cRGD) and monoclonal antibodies (mAbs) targeted for individual integrins have been developed as potential therapeutic drugs for the treatment of several diseases. We showed that a cRGD peptide targeted for alpha(v)beta(3) was internalized in alpha(v)-integrin expressing and nonexpressing melanoma cells by an integrin independent fluid-phase endocytosis pathway that does not alter the number of functional integrin receptors at the cell surface. In contrast, a blocking mAb directed to alpha(v) was internalized by an integrin-dependent endocytosis pathway that reduced the number of functional integrin receptors at the cell surface. We prove that melanoma cells pretreated with the mAb do not readhere to the substrate, whereas cells pretreated with cRGD peptide retain their readhesion capacity. Given the growing importance of RGD peptides, knowledge of these cellular mechanisms is required to improve the development of antiangiogenic and anti-inflammatory drugs.

  2. Modeling the Interaction between Integrin-Binding Peptide (RGD) and Rutile Surface: The Effect of Na+ on Peptide Adsorption

    SciTech Connect

    Wu, Chunya; Skelton, Adam; Chen, Mingjun; Vlcek, Lukas; Cummings, Peter T

    2011-01-01

    The dynamics of a single tripeptide Arg-Gly-Asp (RGD) adsorbing onto negatively charged hydroxylated rutile (110) surface in aqueous solution was studied using molecular dynamics (MD) simulations. The results indicate that the adsorbed Na{sup +} ions play an important role in determining the binding geometry of RGD. With an initial 'horseshoe' configuration, the charged side groups (COO{sup -} and NH{sub 2}) of the peptide are able to interact with the surface through direct hydrogen bonds (H bonds) in the very early stage of adsorption. The Na{sup +} ions approach the positively charged Arg side chain, competing with the Arg side chain for adsorption to the negatively charged hydroxyl oxygen. In coordination with the structural adjustment of the peptide, the Arg residue is driven to detach from the rutile surface. In contrast, the Na+ ions in close proximity to the negatively charged Asp side chain contribute to the binding of the COO{sup -} group on the surface, helping the carboxyl oxygen not involved in COO{sup -}-surface H bonds to orientate toward the hydroxyl hydrogens. Once both carboxyl oxygens form enough H bonds with the hydroxyl hydrogens, the redundant ions move toward a more favorable adsorption site.

  3. Electronic Structure, Dielectric Response, and Surface Charge Distribution of RGD (1FUV) Peptide

    NASA Astrophysics Data System (ADS)

    Adhikari, Puja; Wen, Amy M.; French, Roger H.; Parsegian, V. Adrian; Steinmetz, Nicole F.; Podgornik, Rudolf; Ching, Wai-Yim

    2014-07-01

    Long and short range molecular interactions govern molecular recognition and self-assembly of biological macromolecules. Microscopic parameters in the theories of these molecular interactions are either phenomenological or need to be calculated within a microscopic theory. We report a unified methodology for the ab initio quantum mechanical (QM) calculation that yields all the microscopic parameters, namely the partial charges as well as the frequency-dependent dielectric response function, that can then be taken as input for macroscopic theories of electrostatic, polar, and van der Waals-London dispersion intermolecular forces. We apply this methodology to obtain the electronic structure of the cyclic tripeptide RGD-4C (1FUV). This ab initio unified methodology yields the relevant parameters entering the long range interactions of biological macromolecules, providing accurate data for the partial charge distribution and the frequency-dependent dielectric response function of this peptide. These microscopic parameters determine the range and strength of the intricate intermolecular interactions between potential docking sites of the RGD-4C ligand and its integrin receptor.

  4. Electronic structure, dielectric response, and surface charge distribution of RGD (1FUV) peptide.

    PubMed

    Adhikari, Puja; Wen, Amy M; French, Roger H; Parsegian, V Adrian; Steinmetz, Nicole F; Podgornik, Rudolf; Ching, Wai-Yim

    2014-07-08

    Long and short range molecular interactions govern molecular recognition and self-assembly of biological macromolecules. Microscopic parameters in the theories of these molecular interactions are either phenomenological or need to be calculated within a microscopic theory. We report a unified methodology for the ab initio quantum mechanical (QM) calculation that yields all the microscopic parameters, namely the partial charges as well as the frequency-dependent dielectric response function, that can then be taken as input for macroscopic theories of electrostatic, polar, and van der Waals-London dispersion intermolecular forces. We apply this methodology to obtain the electronic structure of the cyclic tripeptide RGD-4C (1FUV). This ab initio unified methodology yields the relevant parameters entering the long range interactions of biological macromolecules, providing accurate data for the partial charge distribution and the frequency-dependent dielectric response function of this peptide. These microscopic parameters determine the range and strength of the intricate intermolecular interactions between potential docking sites of the RGD-4C ligand and its integrin receptor.

  5. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy.

    PubMed

    Park, Seung-Hwan; Zheng, Jin Hai; Nguyen, Vu Hong; Jiang, Sheng-Nan; Kim, Dong-Yeon; Szardenings, Michael; Min, Jung Hyun; Hong, Yeongjin; Choy, Hyon E; Min, Jung-Joon

    2016-01-01

    Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvβ3, but weakly bound to αvβ3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvβ3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

  6. MS-Monitored Conjugation of Poly(ethylene glycol) Monomethacrylate to RGD Peptides

    PubMed Central

    Bol’shakov, Oleg I.; Akala, Emmanuel O.

    2014-01-01

    Development of biologically active polymers is an active area of research due to their applications in varied and diverse fields of biomedical research: cell adhesion, tissue proliferation, and drug delivery. Recent advances in chemical modification allow fine-tuning of the properties of biomedical polymers to improve their applications: blood circulation half-life, stimuli-responsive degradation, site-specific targeting, drug loading, etc. In this article, convergent synthesis of polymerizable macromonomers bearing a site-specific ligand (RGD peptide) using a low molecular weight MA-poly(ethylene glycols) (PEGs) is presented. The method affords macromonomers useful as the starting materials to produce biomedical polymers. We found matrix assisted laser desorption/ionization mass spectromerty convenient in monitoring the conjugation process via step-by-step following of PEG modification. PMID:24976670

  7. FITC-conjugated cyclic RGD peptides as fluorescent probes for staining integrin αvβ3/αvβ5 in tumor tissues.

    PubMed

    Zheng, Yumin; Ji, Shundong; Czerwinski, Andrzej; Valenzuela, Francisco; Pennington, Michael; Liu, Shuang

    2014-11-19

    This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship

  8. IL-6 Antibody and RGD Peptide Conjugated Poly(amidoamine) Dendrimer for Targeted Drug Delivery of HeLa Cells.

    PubMed

    Mekuria, Shewaye Lakew; Debele, Tilahun Ayane; Chou, Hsiao-Ying; Tsai, Hsieh-Chih

    2016-01-14

    In this study, PAMAM dendrimer (G4.5) was conjugated with two targeting moieties, IL-6 antibody and RGD peptide (G4.5-IL6 and G4.5-RGD conjugates). Doxorubicin anticancer drug was physically loaded onto G4.5-IL6 and G4.5-RGD with the encapsulation efficiency of 51.3 and 30.1% respectively. The cellular internalization and uptake efficiency of G4.5-IL6/DOX and G4.5-RGD/DOX complexes was observed and compared by confocal microscopy and flow cytometry using HeLa cells, respectively. The lower IC50 value of G4.5-IL6/DOX in comparison to G4.5-RGD/DOX is indication that higher drug loading and faster drug release rate corresponded with greater cytotoxicity. The cytotoxic effect was further verified by increment in late apoptotic/necrotic cells due to delivery of drug through receptor-mediated endocytosis. On the basis of these results, G4.5-IL6 is a better suited carrier for targeted drug delivery of DOX to cervical cancer cells.

  9. Dual-functionalized liposomal delivery system for solid tumors based on RGD and a pH-responsive antimicrobial peptide

    PubMed Central

    Zhang, Qianyu; Lu, Libao; Zhang, Li; Shi, Kairong; Cun, Xingli; Yang, Yuting; Liu, Yayuan; Gao, Huile; He, Qin

    2016-01-01

    [D]-H6L9, as a pH-responsive anti-microbial peptide (AMP), has been evidenced by us to be an excellent choice in tumor microenvironment-responsive delivery as it could render liposomes responsive to the acidified tumor microenvironment. However, [D]-H6L9-modified liposomes could not actively target to tumor area. Therefore, integrin αvβ3-targeted peptide RGD was co-modified with [D]-H6L9 onto liposomes [(R + D)-Lip] for improved tumor delivery efficiency. Under pH 6.3, (R + D)-Lip could be taken up by C26 cells and C26 tumor spheroids (integrin αvβ3-positive) with significantly improved efficiency compared with other groups, which was contributed by both RGD and [D]-H6L9, while RGD did not increase the cellular uptake performance on MCF-7 cells (integrin αvβ3-negative). Results showed that RGD could decrease cellular uptake of (R + D)-Lip while [D]-H6L9 could increase it, implying the role of both RGD and [D]-H6L9 in cellular internalization of (R + D)-Lip. On the other hand, (R + D)-Lip could escape the entrapment of lysosomes. PTX-loaded (R + D)-Lip could further increase the cellular toxicity against C26 cells compared with liposomes modified only with RGD and [D]-H6L9 respectively, and achieve remarkable tumor inhibition effect on C26 tumor models. PMID:26842655

  10. Antiadhesive polymer brush coating functionalized with antimicrobial and RGD peptides to reduce biofilm formation and enhance tissue integration.

    PubMed

    Muszanska, Agnieszka K; Rochford, Edward T J; Gruszka, Agnieszka; Bastian, Andreas A; Busscher, Henk J; Norde, Willem; van der Mei, Henny C; Herrmann, Andreas

    2014-06-09

    This paper describes the synthesis and characterization of polymer-peptide conjugates to be used as infection-resistant coating for biomaterial implants and devices. Antiadhesive polymer brushes composed of block copolymer Pluronic F-127 (PF127) were functionalized with antimicrobial peptides (AMP), able to kill bacteria on contact, and arginine-glycine-aspartate (RGD) peptides to promote the adhesion and spreading of host tissue cells. The antiadhesive and antibacterial properties of the coating were investigated with three bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. The ability of the coating to support mammalian cell growth was determined using human fibroblast cells. Coatings composed of the appropriate ratio of the functional components: PF127, PF127 modified with AMP, and PF127 modified with RGD showed good antiadhesive and bactericidal properties without hampering tissue compatibility.

  11. Degradable and biocompatible nanoparticles decorated with cyclic RGD peptide for efficient drug delivery to hepatoma cells in vitro.

    PubMed

    Loyer, Pascal; Bedhouche, Wahib; Huang, Zhi Wei; Cammas-Marion, Sandrine

    2013-10-01

    Amphiphilic derivatives of poly(benzyl malate) were synthesized and characterized with the aim of being used as degradable and biocompatible building blocks for the design of functional nanoparticles (NPs). An anti-cancer model drug, doxorubicin, has been successfully encapsulated into the prepared NPs and its release profile has been evaluated in water and in culture medium. NPs bearing biotin molecules were prepared either for site-specific drug delivery via the targeting of biotin receptors overexpressed on the surface of several cancer cells, or for grafting biotinylated cyclic RGD peptide onto their surface using the strong and highly specific interactions between biotin and the streptavidin protein. We have shown that this binding did not affect dramatically the physico-chemical properties of the corresponding NPs. Cyclic RGD grafted fluorescent NPs were more efficiently uptaken by the HepaRG hepatoma cells than biotinylated fluorescent NPs. Furthermore, the targeting of HepaRG hepatoma cells with NPs bearing cyclic RGD was very efficient and much weaker for HeLa and HT29 cell lines confirming that cyclic RGD is a suitable targeting agent for liver cells. Our results also provide a new mean for rapid screening of short hepatotropic peptides in order to design NPs showing specific liver targeting properties.

  12. Enhanced anticancer efficacy of paclitaxel through multistage tumor-targeting liposomes modified with RGD and KLA peptides

    PubMed Central

    Sun, Jiawei; Jiang, Lei; Lin, Yi; Gerhard, Ethan Michael; Jiang, Xuehua; Li, Li; Yang, Jian; Gu, Zhongwei

    2017-01-01

    Mitochondria serve as both “energy factories” and “suicide weapon stores” of cells. Targeted delivery of cytotoxic drugs to the mitochondria of tumor cells and tumor vascular cells is a promising strategy to improve the efficacy of chemotherapy. Here, multistage tumor-targeting liposomes containing two targeted peptide-modified lipids, cRGD-PEG2000-DSPE and KLA-PEG2000-DSPE, were developed for encapsulation of the anticancer drug paclitaxel (PTX, RGD-KLA/PTX-Lips). Compared with Taxol (free PTX), RGD/PTX-Lips and KLA/PTX-Lips, the half-maximal inhibitory concentration (IC50) value of RGD-KLA/PTX-Lips in vitro was 1.9-, 36.7- and 22.7-fold lower with 4T1 cells, respectively, because of higher levels of cellular uptake. Similar results were also observed with human umbilical vascular endothelial cells (HUVECs). An apoptosis assay showed that the total apoptotic ratio of RGD-KLA/PTX-Lips was the highest because of the mitochondria-targeted drug delivery and the activation of mitochondrial apoptosis pathways, as evidenced by visible mitochondrial localization, decreased mitochondrial membrane potential, release of cytochrome c and increased activities of caspase-9 and caspase-3. The strongest tumor growth inhibition (TGI; 80.6%) and antiangiogenesis effects without systemic toxicity were also observed in RGD-KLA/PTX-Lip-treated 4T1 tumor xenograft BALB/c mice. In conclusion, these multistage tumor-targeting liposomes represent a promising anticancer drug delivery system (DDS) capable of maximizing anticancer therapeutic efficacy and minimizing systemic toxicity. PMID:28280323

  13. Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates.

    PubMed

    Wang, Ke; Zhang, Xiaofeng; Liu, Yang; Liu, Chang; Jiang, Baohong; Jiang, Yanyan

    2014-10-01

    Tumor-penetrating peptide, iRGD (internalizing RGD, CRGDK/RGPD/EC) with the similar affinity to αv integrins as conventional RGD cyclopeptide could enhance the tumor penetrability of drugs by binding to neuropilin-1 (NRP-1) that over-expressed on both angiogenic blood vessels and tumor cells. Comparing with our previous study, in which a RGD cyclopeptide (RGDyC) was bound to PEGylated polyamidoamine (PAMAM) dendrimer with doxorubicin (DOX) by acid-sensitive cis-aconityl linkage (PEG-PAMAM-cis-aconityl-DOX, PPCD), the present study selected iRGD instead of previous RGD to produce iRGD-PPCD conjugate. The effect of iRGD-mediated PPCD on tumor penetration was compared with the conventional RGD ones via administration of RGDs-modified PPCD (iRGD/RGDs-PPCD) and co-administration of RGDs and PPCD (iRGD/RGD + PPCD). C6 cells were selected as the cell model owing to the highest expression of αv integrins and NRP-1 among four tumor cell lines. In vitro cytotoxicity and cellular uptake showed no significant difference between RGD-PPCD and iRGD-PPCD, but glioma spheroid penetration study showed that RGD-PPCD, iRGD-PPCD and iRGD + PPCD penetrated into C6 spheroids with a depth of 115 μm, 144 μm and 150 μm, respectively, indicating that the iRGD-mediated PPCD delivery system had a stronger penetrating ability than the RGD ones. In vivo results also demonstrated the superiority of iRGD system over RGD ones. After systemic administration, iRGD-mediated PPCD increased tumor vascular permeability, decreased tumor vascular density and average vascular diameter. Correspondingly, the iRGD system exhibited stronger penetration ability, higher accumulation in brain tumor. The median survival time of iRGD + PPCD, iRGD-PPCD and RGD-PPCD treatment groups were 61, 57.5 and 43.5 days. The present findings strongly suggested that the iRGD-mediated drug delivery system could significantly improve the efficacy of tumor therapy through enhancing tumor accumulation and penetration as

  14. Impact of Multiple Negative Charges on Blood Clearance and Biodistribution Characteristics of 99mTc-Labeled Dimeric Cyclic RGD Peptides

    PubMed Central

    2015-01-01

    This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of 99mTc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD2 and HYNIC-P6D-RGD2 were prepared by reacting P6G-RGD2 and P6D-RGD2, respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC50 values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against 125I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [99mTc(HYNIC-P6G-RGD2)(tricine)(TPPTS)] (99mTc-P6G-RGD2) and [99mTc(HYNIC-P6D-RGD2)(tricine)(TPPTS)] (99mTc-P6D-RGD2) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37–110 GBq/μmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between 99mTc-P6D-RGD2 and 99mTc-P6G-RGD2 was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for 99mTc-P6D-RGD2 (4.71 ± 1.00%ID/g) was ∼5× higher than that of 99mTc-P6G-RGD2 (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. 99mTc-P6D-RGD2 had much lower tumor uptake (2.20–3.11%ID/g) than 99mTc-P6G-RGD2 (7.82–9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD2 and HYNIC-P6G-RGD2 shared a similar integrin αvβ3 binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of 99mTc-P6D-RGD2. Despite its low uptake in U87MG tumors, the tumor uptake of 99mTc-P6D-RGD2 was integrin αvβ3-specific. SPECT/CT studies were performed using 99mTc-P6G-RGD2 in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of 99mTc-P6G-RGD2, and its tumor uptake depends on the integrin αvβ3 expression levels on tumor cells and neovasculature. It was concluded that

  15. Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility.

    PubMed

    Wang, Yan-Yan; Lü, Lan-Xin; Shi, Jun-Cai; Wang, Hai-Feng; Xiao, Zhong-Dang; Huang, Ning-Ping

    2011-03-14

    Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.

  16. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

    PubMed

    Kong, Lingdan; Alves, Carla S; Hou, Wenxiu; Qiu, Jieru; Möhwald, Helmuth; Tomás, Helena; Shi, Xiangyang

    2015-03-04

    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

  17. Tumor targeting with RGD peptide ligands-design of new molecular conjugates for imaging and therapy of cancers.

    PubMed

    Garanger, Elisabeth; Boturyn, Didier; Dumy, Pascal

    2007-09-01

    Development of molecular devices endowed with tumor-targeting functions and carrying cytotoxic components should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting their peripheral toxicity. Such molecular vectors can pave the way for the development of new classes of therapeutics, fighting against protagonists of neoplastic development. In line with this concept, peptide ligands containing the Arginine-Glycine-Aspartate (RGD) triad, which display a strong affinity and selectivity to the alpha(V)beta(3) integrin, have been developed to target the tumor-associated cells expressing the alpha (V)beta (3) receptors. Among the validated ligands, the leader compound is the cyclic pentapeptide c[-RGDf(NMe)V-] (Cilengitide) developed by kessler et al. (J. Med. Chem., 1999, 42, 3033-3040). This compound has entered phase II clinical trials as an anti-angiogenic agent. Further studies have been directed to develop molecular conjugates of the parent c[-RGDfK-] with conventional chemotherapeutics or with labels for non-invasive imaging technologies. More recently, multimeric RGD containing compounds have been exploited to improve the targeting potential as well as cell-membrane breaching, through receptor-mediated endocytosis. The latter have been constructed on various scaffolds (polylysines or polyglutamates, liposomes, nanoparticles...). Our group has developed a chemical system combining all these properties where multivalent RGD targeting functions are associated with functional molecules through a cyclopeptide template. The latter represents a relevant non-viral vector for tumor targeting, imaging and therapy. This review describes the considerations for the design of the diverse RGD ligands developed so far and reports an overview of the main applications of these structures in cancer research.

  18. Integrin‐Targeting Fluorescent Proteins: Exploration of RGD Insertion Sites

    PubMed Central

    Sonntag, Michael H.; Schill, Jurgen

    2017-01-01

    Abstract The potential of the fluorescent protein scaffold to control peptide sequence functionality is illustrated by an exploration of fluorescent proteins as novel probes for targeting integrins. A library of fluorescent mCitrine proteins with RGD motifs incorporated at several positions in loops within the protein main chain was generated and characterized. Amino acid mutations to RGD as well as RGD insertions were evaluated: both led to constructs with typical mCitrine fluorescent properties. Screening experiments against four human integrin receptors revealed two strong‐binding constructs and two selective integrin binders. The effect of the site of RGD incorporation illustrates the importance of the protein scaffold on RGD sequence functionality, leading to fluorescent protein constructs with the potential for selective integrin targeting. PMID:28004511

  19. RGD-peptide conjugated inulin-ibuprofen nanoparticles for targeted delivery of Epirubicin.

    PubMed

    Zhang, Luzhong; Li, Guicai; Gao, Ming; Liu, Xin; Ji, Bing; Hua, Ruheng; Zhou, Youlang; Yang, Yumin

    2016-08-01

    Recently, chemotherapy-based polymeric nanoparticles have been extensively investigated for solid tumor treatment. Tumor targeted nanoparticles demonstrated great potential for improved accumulation in the tumor tissue, superior anticancer activity and reduced side effects. Thus, inulin-ibuprofen polymer was synthesized by esterification between inulin and ibuprofen, and RGD targeted epirubicin (EPB) loaded nanoparticles were prepared by the self-assembly of inulin-ibuprofen polymer and in situ encapsulation of EPB. RGD conjugated EPB loaded nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). The EPB release from the nanoparticles showed pH-dependent profile and accelerated by the decreased pH value, which would favor the effective drug delivery in vivo. Intracellular uptake analysis suggested that RGD conjugated nanoparticles could be easily internalized by the cancer cells. In vitro cytotoxicity revealed that RGD conjugated EPB loaded nanoparticles exhibited the better antitumor efficacy compared with non-conjugated nanoparticles. More importantly, RGD conjugated EPB loaded nanoparticles showed superior anticancer effects and reduced toxicity than free EPB and non-conjugated nanoparticles by in vivo antitumor activity, EPB biodistribution and histology analysis.

  20. A Novel Strategy to Improve the Therapeutic Efficacy of Gemcitabine for Non-Small Cell Lung Cancer by the Tumor-Penetrating Peptide iRGD

    PubMed Central

    Li, Ke; Wang, Haiyu; Li, Huizhong; Zheng, Junnian

    2015-01-01

    Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, comprising approximately 75–80% of all lung cancers. Gemcitabine is an approved chemotherapy drug for NSCLC. The objective of this study was to develop a novel strategy to improve the therapeutic efficacy of Gemcitabine for NSCLC by the co-administered iRGD peptide. We showed that the rates of positive expression of αvβ3, αvβ5 and NRP-1 in the A549 cell line were 68.5%, 35.3% and 94.5%, respectively. The amount of Evans Blue accumulated in the tumor of Evans Blue+iRGD group was 2.5 times that of Evans Blue group. The rates of growth inhibition of the tumors of the iRGD group, the Gemcitabine group and the Gemcitabine+iRGD group were 8%, 59.8% and 86.9%, respectively. The results of mechanism studies showed that PCNA expression in the Gemcitabine+iRGD group decreased 71.5% compared with that in Gemcitabine group. The rate of apoptosis in the Gemcitabine+iRGD group was 2.2 time that of the Gemcitabine group. Therefore, the tumor-penetrating Peptide iRGD can enhance the tumor-penetrating ability and therapeutic efficacy of Gemcitabine in the A549 xenograft. The combined application of Gemcitabine with iRGD may be a novel strategy to enhance the clinical therapeutic efficacy of Gemcitabine in patients with NSCLC. PMID:26066322

  1. Pharmacophore refinement of gpIIb/IIIa antagonists based on comparative studies of antiadhesive cyclic and acyclic RGD peptides

    NASA Astrophysics Data System (ADS)

    Müller, Gerhard; Gurrath, Marion; Kessler, Horst

    1994-12-01

    Structurally guided design approaches to low-molecular-weight platelet aggregation antagonists addressing the platelet-associated heterodimeric cell surface receptor gpIIb/IIIa rely on comparative studies of an ensemble of conformationally and biologically characterized compounds, since no high-resolution structure of the receptor system is available. We report a classical indirect and comparative pharmacophore refinement approach based on a series of small cyclic Arg-Gly-Asp (RGD) peptides as gpIIb/IIIa-fibrinogen interaction antagonists. These peptides have previously been investigated as potent and selective tumor cell adhesion inhibitors. The definition of geometrical descriptors classifying the RGD peptide conformations and their subsequent analysis over selected RGD- and RXD-containing protein structures allows for a correlation of distinct structural features for platelet aggregation inhibition. An almost parallel alignment of the Arg and Asp side chains was identified by a vector analysis as being present in all active cyclic hexa-and pentapeptides. This orientation is induced mainly by the constraint of backbone cyclization and is not of any covalent tripeptide-inherent origin, which was rationalized by a 500 ps high-energy MD simulation of a sequentially related linear model peptide. The incorporation of the recognition tripeptide Arg-Gly-Asp into the cyclic peptide templates acted as a filter mechanism, restricting the otherwise free torsional relation of both side chains to a parallel orientation. Based on the derived results, several detailed features of the receptor binding site could be deduced in terms of receptor complementarity. These findings should govern the design of next-generation compounds with enhanced activities. Furthermore, the complementary stereochemical characteristics of the substrate can be used as boundary conditions for pseudoreceptor modelling studies that are capable of reconstructing a hypothetical binding pocket

  2. Radiolabeled Cyclic RGD Peptides as Radiotracers for Imaging Tumors and Thrombosis by SPECT

    PubMed Central

    Zhou, Yang; Chakraborty, Sudipta; Liu, Shuang

    2011-01-01

    The integrin family is a group of transmembrane glycoprotein comprised of 19 α- and 8 β-subunits that are expressed in 25 different α/β heterodimeric combinations on the cell surface. Integrins play critical roles in many physiological processes, including cell attachment, proliferation, bone remodeling, and wound healing. Integrins also contribute to pathological events such as thrombosis, atherosclerosis, tumor invasion, angiogenesis and metastasis, infection by pathogenic microorganisms, and immune dysfunction. Among 25 members of the integrin family, the αvβ3 is studied most extensively for its role of tumor growth, progression and angiogenesis. In contrast, the αIIbβ3 is expressed exclusively on platelets, facilitates the intercellular bidirectional signaling (“inside-out” and “outside-in”) and allows the aggregation of platelets during vascular injury. The αIIbβ3 plays an important role in thrombosis by its activation and binding to fibrinogen especially in arterial thrombosis due to the high blood flow rate. In the resting state, the αIIbβ3 on platelets does not bind to fibrinogen; on activation, the conformation of platelet is altered and the binding sites of αIIbβ3 are exposed for fibrinogen to crosslink platelets. Over the last two decades, integrins have been proposed as the molecular targets for diagnosis and therapy of cancer, thrombosis and other diseases. Several excellent review articles have appeared recently to cover a broad range of topics related to the integrin-targeted radiotracers and their nuclear medicine applications in tumor imaging by single photon emission computed tomography (SPECT) or a positron-emitting radionuclide for positron emission tomography (PET). This review will focus on recent developments of αvβ3-targeted radiotracers for imaging tumors and the use of αIIbβ3-targeted radiotracers for thrombosis imaging, and discuss different approaches to maximize the targeting capability of cyclic RGD peptides

  3. Robust Therapeutic Efficacy of Matrix Metalloproteinase-2-Cleavable Fas-1-RGD Peptide Complex in Chronic Inflammatory Arthritis

    PubMed Central

    Sa, Keum Hee; Sung, Shijin; Park, Jae Yong; Jo, Dong-Gyu; Park, Jae Hyung; Kim, In San; Kang, Young Mo

    2016-01-01

    Objective Therapeutic agents that are transformable via introducing cleavable linkage by locally enriched MMP-2 within inflamed synovium would enhance therapeutic efficacy on chronic inflammatory arthritis. Transforming growth factor-β-inducible gene-h3 (βig-h3), which consists of four fas-1 domains and an Arg-Gly-Asp (RGD) motif, intensifies inflammatory processes by facilitating adhesion and migration of fibroblast-like synoviocyte in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to investigate whether a MMP-2-cleavable peptide complex consisting of a fas-1 domain and an RGD peptide blocks the interaction between βig-h3 and resident cells and leads to the amelioration of inflammatory arthritis. Methods We designed βig-h3-derivatives, including the fourth fas-1 domain truncated for H1 and H2 sequences of mouse (MFK00) and MMP-2-cleavable peptide complex (MFK902). MMP-2 selectivity was examined by treatment with a series of proteases. MFK902 efficacy was determined by the adhesion and migration assay with NIH3T3 cells in vitro and collagen-induced arthritis (CIA) model using male DBA/1J mice in vivo. The mice were treated intraperitoneally with MFK902 at different dosages. Results MFK902 was specifically cleaved by active MMP-2 in a concentration-dependent manner, and βig-h3-mediated adhesion and migration were more effectively inhibited by MFK902, compared with RGD or MFK00 peptides. The arthritis activity of murine CIA, measured by clinical arthritis index and incidence of arthritic paws, was significantly ameliorated after treatment with all dosages of MFK902 (1, 10, and 30 mg/kg). MFK902 ameliorated histopathologic deterioration and reduced the expression of inflammatory mediators simultaneously with improvement of clinical features. In addition, a favorable safety profile of MFK902 was demonstrated in vivo. Conclusion The present study revealed that MMP-2-cleavable peptide complex based on βig-h3 structure is a potent and safe

  4. Integrin αvβ3 as a Promising Target to Image Neoangiogenesis Using In-House Generator-Produced Positron Emitter (68)Ga-Labeled DOTA-Arginine-Glycine-Aspartic Acid (RGD) Ligand.

    PubMed

    Vatsa, Rakhee; Bhusari, Priya; Kumar, Sunil; Chakraborty, Sudipta; Dash, Ashutosh; Singh, Gurpreet; Dhawan, Devinder Kumar; Shukla, Jaya; Mittal, Bhagwant Rai

    2015-06-01

    For the growth and spread of a tumor beyond 2 mm, angiogenesis plays a crucial role, and association of various integrins with angiogenesis is evidential. The aim of the study was radiolabeling of DOTA-chelated RGD (arginine-glycine-aspartic acid) peptide with (68)Ga for PET imaging in locally advanced breast carcinoma. DOTA-RGD was incubated with (68)GaCl3, eluted in 0.05 m HCl. Elution volume, peptide amount, and reaction pH were studied. Radio-ITLC, gas chromatography, endotoxin, and sterility testing were performed. Serial (n=3) and whole-body (n=2) PET/CT imaging was done on patients post i.v. injection of 111-185 MBq of (68)Ga-DOTA-RGD. Maximum radiolabeling yield was achieved with 3 mL elution volume of 15-20 μg peptide at pH 3.5-4.0 with 10 minutes of incubation at 95°C. Product samples were sterile having 99.5% radiochemical purity with residual ethanol content and endotoxins in injectable limits. Intense radiotracer uptake was noticed in the tumor with SUVmax 15.3 at 45 minutes in serial images. Physiological radiotracer uptake was seen in the liver, spleen, ventricles, and thyroid with excretion through the kidneys. The authors concluded that (68)Ga-DOTA-RGD has the potential for imaging α,vβ3 integrin-expressing tumors.

  5. [68Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold—synthesis and evaluation

    PubMed Central

    Knetsch, Peter A.; Zhai, Chuangyan; Rangger, Christine; Blatzer, Michael; Haas, Hubertus; Kaeopookum, Piriya; Haubner, Roland; Decristoforo, Clemens

    2015-01-01

    Over the last years Gallium-68 (68Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). 68Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for 68Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based 68Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and 68Ga-radiolabeling of a trimeric FSC-RGD conjugate, [68Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with 68Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [68Ga]FSC-(RGD)3 was prepared with high radiochemical yield (> 98%). Distribution coefficient was − 3.6 revealing a hydrophilic character, and an IC50 value of 1.8 ± 0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [68Ga]FSC-(RGD)3 was stable in PBS (pH 7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37 °C for 2 hours. Biodistribution assay confirmed

  6. [(68)Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold-synthesis and evaluation.

    PubMed

    Knetsch, Peter A; Zhai, Chuangyan; Rangger, Christine; Blatzer, Michael; Haas, Hubertus; Kaeopookum, Piriya; Haubner, Roland; Decristoforo, Clemens

    2015-02-01

    Over the last years Gallium-68 ((68)Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). (68)Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for (68)Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based (68)Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and (68)Ga-radiolabeling of a trimeric FSC-RGD conjugate, [(68)Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with (68)Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [(68)Ga]FSC-(RGD)3 was prepared with high radiochemical yield (>98%). Distribution coefficient was -3.6 revealing a hydrophilic character, and an IC50 value of 1.8±0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [(68)Ga]FSC-(RGD)3 was stable in PBS (pH7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37°C for 2hours. Biodistribution assay

  7. Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis.

    PubMed

    Kato, Hirotomo; Gomez, Eduardo A; Fujita, Megumi; Ishimaru, Yuka; Uezato, Hiroshi; Mimori, Tatsuyuki; Iwata, Hiroyuki; Hashiguchi, Yoshihisa

    2015-05-01

    Sequence analysis of the Lutzomyia (Lu.) ayacuchensis salivary gland cDNA library identified a short peptide containing an RGD (Arg-Gly-Asp) sequence flanked by two cysteine residues in the C-terminal end as the most abundant transcript. In the present study, a recombinant protein of the RGD-containing peptide, designated ayadualin, was expressed in Escherichia coli and its activity was characterized. Ayadualin inhibited both collagen and ADP-induced platelet aggregations by interfering with the binding of integrin αIIbβ3 to fibrinogen. The RGD sequence and cysteine residues located on both sides of the RGD sequence were essential for the inhibitory action. Moreover, ayadualin efficiently inhibited the intrinsic blood coagulation pathway irrespective of the RGD sequence. Measuring the enzymatic activity of coagulation factors using chromogenic substrates revealed that ayadualin efficiently inhibited factor XIIa (FXIIa) activity in a dose-dependent manner. In addition, pre-incubation of ayadualin with FXII inhibited FXIIa activity, while activated FXIIa was not affected by ayadualin, indicating that ayadualin inhibits the activation of FXII, but not enzymatic activity of FXIIa. These results indicated that ayadualin plays an important role in the blood feeding of Lu. ayacuchensis by inhibiting host hemostasis via dual mechanisms.

  8. Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion

    PubMed Central

    Pallarola, Diego; Bochen, Alexander; Boehm, Heike; Rechenmacher, Florian; Sobahi, Tariq R; Spatz, Joachim P; Kessler, Horst

    2014-01-01

    The interaction of specific surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand's spacer chemistry and length reveal significant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization. PMID:25810710

  9. High affinity recognition of a Phytophthora protein by Arabidopsis via an RGD motif.

    PubMed

    Senchou, V; Weide, R; Carrasco, A; Bouyssou, H; Pont-Lezica, R; Govers, F; Canut, H

    2004-02-01

    The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [125I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53-56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp56 into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.

  10. Sialylation transmogrifies human breast and pancreatic cancer cells into 3D multicellular tumor spheroids using cyclic RGD-peptide induced self-assembly.

    PubMed

    Akasov, Roman; Haq, Sabah; Haxho, Fiona; Samuel, Vanessa; Burov, Sergey V; Markvicheva, Elena; Neufeld, Ronald J; Szewczuk, Myron R

    2016-10-04

    Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix-free spheroids from breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD platform of cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK (TPP)). MCF-7 and MCF-7 TMX cells formed tight spheroids both in the classical agarose-and RGD-based platforms while all PANC1 cells formed loose aggregates. Using lectin histochemistry staining, sialidase assay, neuraminidase (Vibrio cholerae) and oseltamivir phosphate (OP) neuraminidase inhibitor treatments, MCF-7 and PANC1 cells and their drug-resistant variants expressed different sialic acid (SA) content on their cell surfaces. α-2,3- and α-2,6-sialic acid surface residues facilitated spheroid formation under cyclo-RGDfK(TPP)-induced self-assembly. Pretreatment with α-2,3- SA specific Maackia amurensis (MAL-II) lectin, α-2,6-SA specific Sambucus nigra (SNA) lectin, and exogenous α-2,6-SA specific neuraminidase (Vibrio cholerae) dose-dependently reduced spheroid volume. OP enhanced cell aggregation and compaction forming spheroids. PANC1 and MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significantly higher levels of α-2,3- SA over α-2,6-SA. MCF-7 spheroids also expressed a high α-2,3-SA to α-2,6-SA ratio. These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors.

  11. Cyclic RGD peptide-modified liposomal drug delivery system for targeted oral apatinib administration: enhanced cellular uptake and improved therapeutic effects

    PubMed Central

    Song, Zhiwang; Lin, Yun; Zhang, Xia; Feng, Chan; Lu, Yonglin; Gao, Yong; Dong, Chunyan

    2017-01-01

    Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin αvβ3 and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer. PMID:28331317

  12. Galloyl-RGD as a new cosmetic ingredient

    PubMed Central

    2014-01-01

    Background The cosmetics market has rapidly increased over the last years. For example, in 2011 it reached 242.8 billion US dollars, which was a 3.9% increase compared to 2010. There have been many recent trials aimed at finding the functional ingredients for new cosmetics. Gallic acid is a phytochemical derived from various herbs, and has anti-fungal, anti-viral, and antioxidant properties. Although phytochemicals are useful as cosmetic ingredients, they have a number of drawbacks, such as thermal stability, residence time in the skin, and permeability through the dermal layer. To overcome these problems, we considered conjugation of gallic acid with a peptide. Results We synthesized galloyl-RGD, which represents a conjugate of gallic acid and the peptide RGD, purified it by HPLC and characterized by MALDI-TOF with the aim of using it as a new cosmetic ingredient. Thermal stability of galloyl-RGD was tested at alternating temperatures (consecutive 4°C, 20°C, or 40°C for 8 h each) on days 2, 21, 41, and 61. Galloyl-RGD was relatively safe to HaCaT keratinocytes, as their viability after 48 h incubation with 500 ppm galloyl-RGD was 93.53%. In the group treated with 50 ppm galloyl-RGD, 85.0% of free radicals were removed, whereas 1000 ppm galloyl-RGD suppressed not only L-DOPA formation (43.8%) but also L-DOPA oxidation (54.4%). Conclusions Galloyl-RGD is a promising candidate for a cosmetic ingredient. PMID:25103826

  13. Modification of the adhesive properties of collagen by covalent grafting with RGD peptides.

    PubMed

    Myles, J L; Burgess, B T; Dickinson, R B

    2000-01-01

    Collagen, either alone or in combination with other materials, is an important natural biomaterial that is used in a variety of tissue-engineering applications. Cell adhesion and migration of cells within collagen-based biomaterials may be controlled by modifying the adhesive properties of collagen. Furthermore, spatially controlling the adhesiveness of the collagen may allow controlled localization or redistribution of cells. A method is presented for covalently coupling peptides that contain the well-characterized arginine-glycine-aspartic acid adhesion sequence directly to type I collagen monomers prior to fibrillogenesis. A heterobifunctional coupling agent was used to create stable amide and disulfide bonds with the lysine residues of the collagen monomers and the cysteine termini of the peptide molecules, respectively. The degree of conjugation could be controlled by changing the reaction conditions (ratios of reactants added and the length of incubation). The microstructure and gelation times of gels composed of covalently modified collagen were similar to those of unmodified gels. Cell adhesion on adsorbed monolayers of modified collagen was quantified using a well-established clonal cell line (K1735 murine melanoma). Cell adhesion was found to increase with both increasing degree of conjugation and increasing ratio of modified to unmodified collagen.

  14. RGD functionalized polymeric nanoparticles targeting periodontitis epithelial cells for the enhanced treatment of periodontitis in dogs.

    PubMed

    Yao, Wenxin; Xu, Peicheng; Zhao, Jingjing; Ling, Li; Li, Xiaoxia; Zhang, Bo; Cheng, Nengneng; Pang, Zhiqing

    2015-11-15

    Long term retention of antimicrobials with effective drug concentration in gingival crevicular fluid (GCF) is of vital importance for the treatment of chronic periodontitis. In this study, a novel epithelial cell-targeting nanoparticle drug delivery system by conjugating minocycline-loaded poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles (NP-MIN) with RGD peptide were developed and administrated locally for targeting periodontitis epithelial cells and enhancing the treatment of periodontitis in dogs. Biodegradable NP-MIN was made with an emulsion/solvent evaporation technique. RGD peptide was conjugated to the surface of nanoparticles via Maleimide group reaction with hydrosulfide in RGD peptide (RGD-NP-MIN). Transmission electron microscopy examination and dynamic light scattering results revealed that RGD-NP-MIN had a sphere shape, with a mean diameter around 106nm. In vitro release of minocycline from RGD-NP-MIN showed that RGD modification did not change the remarkable sustained releasing characteristic of NP-MIN. To elucidate the interaction of RGD-NP and epithelial cells, RGD-NP binding, uptake and cellular internalization mechanisms by calu-3 cells were investigated. It was shown RGD modification significantly enhanced nanoparticles binding and uptake by Calu-3 cells, and RGD-NP uptake was an energy-dependent process through receptor-mediated endocytosis. Both clathrin-associated endocytosis and caveolae-dependent endocytosis pathway were involved in the RGD-NP uptake, and the intracellular transport of RGD-NP was related to lysosome and Golgi apparatus. Finally, in vivo pharmacokinetics of minocycline in the periodontal pockets and anti-periodontitis effects of RGD-NP-MIN on periodontitis-bearing dogs were evaluated. After local administration of RGD-NP-MIN, minocycline concentration in gingival crevicular fluid decreased slowly and maintained an effective drug concentration for a longer time than that of NP-MIN. Anti-periodontitis effects

  15. Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

    PubMed

    Frutos, S; Jordan, J B; Bio, M M; Muir, T W; Thiel, O R; Vila-Perelló, M

    2016-10-12

    An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

  16. Sulfonation of Tyrosine as a Method to Improve Biodistribution of Peptide-Based Radiotracers: Novel (18)F-Labelled Cyclic RGD Analogues.

    PubMed

    Haskali, Mohammad Baqir; Denoyer, Delphine; Noonan, Wayne; Cullinane, Carleen; Rangger, Christine; Pouliot, Normand; Haubner, Roland; Roselt, Peter D; Hicks, Rodney J; Hutton, Craig A

    2017-02-13

    The labeling of peptides with positron emitting radionuclides has long held the promise of a wide range of PET agents possessing high affinity and selectivity. Not surprisingly, controlling the biodistribution of these agents has proven to be a major challenge in their successful application. Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[18F]fluoropropionate and the biodistribution of the radiolabeled peptides was compared with that of their non-sulfonated, clinically relevant counterparts, [18F]GalactoRGD and [18F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da towards the MW, compared with 189 Da for both the 'Galacto' and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabelled peptides.

  17. RGD-based PET tracers for imaging receptor integrin αv β3 expression.

    PubMed

    Cai, Hancheng; Conti, Peter S

    2013-05-15

    Positron emission tomography (PET) imaging of receptor integrin αv β3 expression may play a key role in the early detection of cancer and cardiovascular diseases, monitoring disease progression, evaluating therapeutic response, and aiding anti-angiogenic drugs discovery and development. The last decade has seen the development of new PET tracers for in vivo imaging of integrin αv β3 expression along with advances in PET chemistry. In this review, we will focus on the radiochemistry development of PET tracers based on arginine-glycine-aspartic acid (RGD) peptide, present an overview of general strategies for preparing RGD-based PET tracers, and review the recent advances in preparations of (18) F-labeled, (64) Cu-labeled, and (68) Ga-labeled RGD tracers, RGD-based PET multivalent probes, and RGD-based PET multimodality probes for imaging receptor integrin αv β3 expression.

  18. Sialylation transmogrifies human breast and pancreatic cancer cells into 3D multicellular tumor spheroids using cyclic RGD-peptide induced self-assembly

    PubMed Central

    Akasov, Roman; Haq, Sabah; Haxho, Fiona; Samuel, Vanessa; Burov, Sergey V.; Markvicheva, Elena; Neufeld, Ronald J.; Szewczuk, Myron R.

    2016-01-01

    Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix-free spheroids from breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD platform of cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK (TPP)). MCF-7 and MCF-7 TMX cells formed tight spheroids both in the classical agarose-and RGD-based platforms while all PANC1 cells formed loose aggregates. Using lectin histochemistry staining, sialidase assay, neuraminidase (Vibrio cholerae) and oseltamivir phosphate (OP) neuraminidase inhibitor treatments, MCF-7 and PANC1 cells and their drug-resistant variants expressed different sialic acid (SA) content on their cell surfaces. α-2,3- and α-2,6-sialic acid surface residues facilitated spheroid formation under cyclo-RGDfK(TPP)-induced self-assembly. Pretreatment with α-2,3- SA specific Maackia amurensis (MAL-II) lectin, α-2,6-SA specific Sambucus nigra (SNA) lectin, and exogenous α-2,6-SA specific neuraminidase (Vibrio cholerae) dose-dependently reduced spheroid volume. OP enhanced cell aggregation and compaction forming spheroids. PANC1 and MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significantly higher levels of α-2,3- SA over α-2,6-SA. MCF-7 spheroids also expressed a high α-2,3-SA to α-2,6-SA ratio. These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors. PMID:27608845

  19. Peptide-22 and Cyclic RGD Functionalized Liposomes for Glioma Targeting Drug Delivery Overcoming BBB and BBTB.

    PubMed

    Chen, Cuitian; Duan, Ziqing; Yuan, Yan; Li, Ruixiang; Pang, Liang; Liang, Jianming; Xu, Xinchun; Wang, Jianxin

    2017-02-22

    Chemotherapy outcomes for the treatment of glioma remain unsatisfied due to the inefficient drug transport across BBB/BBTB and poor drug accumulation in the tumor site. Nanocarriers functionalized with different targeting ligands are considered as one of the most promising alternatives. However, few studies were reported to compare the targeting efficiency of the ligands and develop nanoparticles to realize BBB/BBTB crossing and brain tumor targeting simultaneously. In this study, six peptide-based ligands (Angiopep-2, T7, Peptide-22, c(RGDfK), D-SP5 and Pep-1), widely used for brain delivery, were selected to decorate liposomes, respectively, so as to compare their targeting ability to BBB or BBTB. Based on the in vitro cellular uptake results on BCECs and HUVECs, Peptide-22 and c(RGDfK) were picked to construct a BBB/BBTB dual-crossing, glioma-targeting liposomal drug delivery system c(RGDfK)/Pep-22-DOX-LP. In vitro cellular uptake demonstrated that the synergetic effect of c(RGDfK) and Peptide-22 could significantly increase the internalization of liposomes on U87 cells. In vivo imaging further verified that c(RGDfK)/Pep-22-LP exhibited higher brain tumor distribution than single ligand modified liposomes. The median survival time of glioma-bearing mice treated with c(RGDfK)/Pep-22-DOX-LP (39.5 days) was significantly prolonged than those treated with free doxorubicin or other controls. In conclusion, the c(RGDfK) and Peptide-22 dual-modified liposome was constructed based on the targeting ability screening of various ligands. The system could effectively overcome BBB/BBTB barriers, target to tumor cells and inhibit the growth of glioma, which proved its potential for improving the efficacy of chemotherapeutics for glioma therapy.

  20. Cyclic RGD conjugated poly(ethylene glycol)-co-poly(lactic acid) micelle enhances paclitaxel anti-glioblastoma effect.

    PubMed

    Zhan, Changyou; Gu, Bing; Xie, Cao; Li, Jin; Liu, Yu; Lu, Weiyue

    2010-04-02

    The use of glioblastoma-targeted drug delivery system facilitates efficient delivery of chemotherapeutic agents to malignant gliomas in the central nervous system while minimizing high systemic doses associated with debilitating toxicities. To employ the high binding affinity of a cyclic RGD peptide (c(RGDyK), cyclic Arginine-Glycine-Aspartic acid-D-Tyrosine-Lysine) with integrin alpha(v)beta(3) over-expressed on tumor neovasculature and U87MG glioblastoma cells, we prepared paclitaxel-loaded c(RGDyK)-Poly(ethylene glycol)-block-poly(lactic acid) micelle (c(RGDyK)-PEG-PLA-PTX). In vitro physicochemical characterization of these novel micelles showed satisfactory encapsulated efficiency, loading capacity and size distribution. In vitro cytotoxicity studies proved that the presence of c(RGDyK) enhanced the anti-glioblastoma cell cytotoxic efficacy by 2.5 folds. The binding affinity of c(RGDyK)-PEG-PLA micelle with U87MG cells was also investigated. The competitive binding IC(50) value of c(RGDyK)-PEG-PLA micelle was 26.30 nM, even lower than that of c(RGDyK) (56.23 nM). In U87MG glioblastoma-bearing nude mice model, biodistribution of (125)I-radiolabeled c(RGDyK)-PEG-PLA or DiR encapsulated micelles and anti-glioblastoma pharmacological effect was investigated after intravenous administration. c(RGDyK)-PEG-PLA micelle accumulated in the subcutaneous and intracranial tumor tissue, and when loaded with PTX (c(RGDyK)-PEG-PLA-PTX), exhibited the strongest tumor growth inhibition among the studied paclitaxel formulations. The anti-glioblastoma effect of c(RGDyK)-PEG-PLA-PTX micelle was also reflected in the median survival time of mice bearing intracranial U87MG tumor xenografts where the median survival time of c(RGDyK)-PEG-PLA-PTX micelle-treated mice (48 days) was significantly longer than that of mice treated with PEG-PLA-PTX micelle (41.5 days), Taxol (38.5 days) or saline (34 days). Therefore, our results suggested that c(RGDyK)-PEG-PLA micelle may be a potential

  1. Effect of RGD-functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

    PubMed Central

    Gribova, Varvara; Gauthier-Rouvière, Cécile; Albigès-Rizo, Corinne; Auzely-Velty, Rachel; Picart, Catherine

    2014-01-01

    Skeletal muscle tissue engineering holds promise for the replacement of muscle due to an injury and for the treatment of muscle diseases. Although RGD substrates have been widely explored in tissue engineering, there is no study aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work, we use polyelectrolyte multilayer films made of poly(L-lysine) (PLL) and poly(L-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increased film stiffness could both induce cell adhesion, but integrins involved in adhesion were different in case of soft and stiff films. Moreover, soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation while the stiff PLL/PGA films significantly induced cell migration, proliferation and inhibited myogenic differentiation. The ROCK kinase was found to be involved in myoblast response to the different films. Indeed, its inhibition was sufficient to rescue the differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on mechanical and biochemical properties of the multilayer films. This study emphasizes the superior advantage of the soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as coating of various polymeric scaffolds for muscle tissue engineering. PMID:23261924

  2. Computational study of the RGD-peptide interactions with perovskite-type BFO-(1 1 1) membranes under aqueous conditions

    NASA Astrophysics Data System (ADS)

    Li, Hai-long; Bian, Liang; Hou, Wen-ping; Dong, Fa-Qin; Song, Mian-Xin; Zhang, Xiao-yan; Wang, Li-sheng

    2016-07-01

    We elucidated a number of facets regarding arginine-glycine-aspartate (RGD)-bismuth ferrite (BFO)-(1 1 1) membrane interactions and reactivity that have previously remained unexplored on a molecular level. Results demonstrate the intra-molecular interaction facilitates a ;horseshoe; structure of RGD adsorbed onto the BFO-(1 1 1) membrane, through the electrostatic (Asp-cation-Fe) and water-bridge (Osbnd H2O and H2Osbnd NH2) interactions. The effect of structural and electron-transfer interactions is attributed to the cation-valences, indicating that the divalent cations are electron-acceptors and the monovalent cations as electron-donors. Notably, the strongly bound Ca2+ ion exerts a ;gluing; effect on the Asp-side-chain, indicating a tightly packed RGD-BFO configuration. Thus, modulating the biological response of BFO-(1 1 1) membrane will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic perovskite-type micro-devices.

  3. Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid-paclitaxel (CLA-PTX) on B16-F10 melanoma.

    PubMed

    Du, Ruo; Zhong, Ting; Zhang, Wei-Qiang; Song, Ping; Song, Wen-Ding; Zhao, Yang; Wang, Chao; Tang, Yi-Qun; Zhang, Xuan; Zhang, Qiang

    2014-01-01

    In the present study, we prepared a novel delivery system of iRGD (CRGDK/RGPD/EC)-modified sterically stabilized liposomes (SSLs) containing conjugated linoleic acid-paclitaxel (CLA-PTX). The anti-tumor effect of iRGD-SSL-CLA-PTX was investigated on B16-F10 melanoma in vitro and in vivo. The in vitro targeting effect of iRGD-modified SSLs was investigated in a real-time confocal microscopic analysis experiment. An endocytosis-inhibition assay was used to evaluate the endocytosis pathways of the iRGD-modified SSLs. In addition, the in vitro cellular uptake and in vitro cytotoxicity of iRGD-SSL-CLA-PTX were evaluated in B16-F10 melanoma cells. In vivo biodistribution and in vivo antitumor effects of iRGD-SSL-CLA-PTX were investigated in B16-F10 tumor-bearing mice. The induction of apoptosis by iRGD-SSL-CLA-PTX was evaluated in tumor-tissue sections. Real-time confocal microscopic analysis results indicated that the iRGD-modified SSLs internalized into B16-F10 cells faster than SSLs. The identified endocytosis pathway of iRGD-modified SSLs indicated that energy- and lipid raft-mediated endocytosis played a key role in the liposomes' cellular uptake. The results of the cellular uptake experiment indicated that the increased cellular uptake of CLA-PTX in the iRGD-SSL-CLA-PTX-treated group was 1.9-, 2.4-, or 2.1-fold compared with that in the CLA-PTX group after a 2-, 4-, or 6-hour incubation, respectively. In the biodistribution test, the CLA-PTX level in tumor tissues from iRGD-SSL-CLA-PTX-treated mice at 1 hour (1.84±0.17 μg/g) and 4 hours (1.17±0.28 μg/g) was 2.3- and 2.0-fold higher than that of CLA-PTX solution at 1 hour (0.79±0.06 μg/g) and 4 hours (0.58±0.04 μg/g). The value of the area under the curve for the first 24 hours in the tumors of iRGD-SSL-CLA-PTX-treated mice was significantly higher than that in the SSL-CLA-PTX and CLA-PTX solution-treated groups (P<0.01). The in vivo antitumor results indicated that iRGD-SSL-CLA-PTX significantly inhibited

  4. RGD Peptides-Conjugated Pluronic Triblock Copolymers Encapsulated with AP-2α Expression Plasmid for Targeting Gastric Cancer Therapy in Vitro and in Vivo.

    PubMed

    Wang, Wei; Liu, Zhimin; Sun, Peng; Fang, Cheng; Fang, Hongwei; Wang, Yueming; Ji, Jiajia; Chen, Jun

    2015-07-17

    Gastric cancer, a high-risk malignancy, is a genetic disease developing from a cooperation of multiple gene mutations and a multistep process. Gene therapy is a novel treatment method for treating gastric cancer. Here, we developed a novel Arg-Gly-Asp (RGD) peptides conjugated copolymers nanoparticles-based gene delivery system in order to actively targeting inhibit the growth of gastric cancer cells. These transcription factor (AP-2α) expression plasmids were also encapsulated into pluronic triblock copolymers nanoparticles which was constituted of poly(ethylene glycol)-block-poly(propylene glycol)- block-poly(ethylene glycol) (PEO-block-PPO-block-PEO, P123). The size, morphology and composition of prepared nanocomposites were further characterized by nuclear magnetic resonance (NMR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). In MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) analysis, these nanocomposites have minor effects on the proliferation of GES-1 cells but significantly decreased the viability of MGC-803, suggesting they own low cytotoxicity but good antitumor activity. The following in vivo evaluation experiments confirmed that these nanocomposites could prevent the growth of gastric cancer cells in the tumor xenograft mice model. In conclusion, these unique RGD peptides conjugated P123 encapsulated AP-2α nanocomposites could selectively and continually kill gastric cancer cells by over-expression of AP-2α in vitro and in vivo; this exhibits huge promising applications in clinical gastric cancer therapy.

  5. Transferred-NOE NMR experiments on intact human platelets: receptor-bound conformation of RGD-peptide mimics.

    PubMed

    Potenza, Donatella; Belvisi, Laura

    2008-01-21

    The aim of this work is to show that transferred-NOE provides useful and detailed information on membrane-bound receptor-ligand interactions in living cells. Here, we study the interaction between intact human platelets and some ligands containing the RGD sequence. Conformational properties of the free and bound pentapeptides are reported.

  6. Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach.

    PubMed

    Lu, X; Lu, D; Scully, M F; Kakkar, V V

    2003-06-01

    Integrins are a family of heterodimeric class I transmembrane receptors, many of which bind to the RGD sequence in adhesive proteins and mediate the adhesive interactions of a variety of cells. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with beta1 and beta3 associated integrins and their potency is at least 500-2000 times higher than short RGD peptides. Structural and functional studies suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined flexible loop. The integrin-binding specificity and selectivity of each of the proteins is controlled by amino acid residues in this loop in close vicinity to the RGD-motif. The review includes an overview of the structure and function of snake-venom integrin antagonists. The ability of these proteins to control platelet aggregation, cell adhesion and ligand binding is compared to that of short linear, cyclic RGD-peptides and RGD-containing proteins and the influence of modulation of amino acid residues flanking the RGD motif is also considered. The review is intended to provide insight into the development of novel inhibitors as drugs.

  7. Borinic acid catalysed peptide synthesis.

    PubMed

    El Dine, Tharwat Mohy; Rouden, Jacques; Blanchet, Jérôme

    2015-11-18

    The catalytic synthesis of peptides is a major challenge in the modern organic chemistry hindered by the well-established use of stoichiometric coupling reagents. Herein, we describe for the first time that borinic acid is able to catalyse this reaction under mild conditions with an improved activity compared to our recently developed thiophene-based boronic acid. This catalyst is particularly efficient for peptide bond synthesis affording dipeptides in good yields without detectable racemization.

  8. Development of pre-implantation porcine embryos cultured within a three-dimensional alginate hydrogel system either conjugated with Arg-Gly-Asp (RGD) peptide or supplemented with secreted phosphoprotein 1 (SPP1)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many uterine specific factors have been shown to be increased within the uterine milieu as the porcine embryo initiates elongation. Secreted phosphoprotein 1 (SPP1) is increased during this time and contains an Arg-Gly-Asp (RGD) peptide sequence that has been shown to bind to cell surface integrins ...

  9. The bioactive acidic serine- and aspartate-rich motif peptide.

    PubMed

    Minamizaki, Tomoko; Yoshiko, Yuji

    2015-01-01

    The organic component of the bone matrix comprises 40% dry weight of bone. The organic component is mostly composed of type I collagen and small amounts of non-collagenous proteins (NCPs) (10-15% of the total bone protein content). The small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a NCP, is considered to play a key role in bone mineralization. SIBLING family of proteins share common structural features and includes the arginine-glycine-aspartic acid (RGD) motif and acidic serine- and aspartic acid-rich motif (ASARM). Clinical manifestations of gene mutations and/or genetically modified mice indicate that SIBLINGs play diverse roles in bone and extraskeletal tissues. ASARM peptides might not be primary responsible for the functional diversity of SIBLINGs, but this motif is suggested to be a key domain of SIBLINGs. However, the exact function of ASARM peptides is poorly understood. In this article, we discuss the considerable progress made in understanding the role of ASARM as a bioactive peptide.

  10. RGD-FasL Induces Apoptosis in Hepatocellular Carcinoma

    PubMed Central

    Liu, Zhongchen; Wang, Juan; Yin, Ping; Qiu, Jinhua; Liu, Ruizhen; Li, Wenzhu; Fan, Xin; Cheng, Xiaofeng; Chen, Caixia; Zhang, Jiakai; Zhuang, Guohong

    2009-01-01

    Despite impressive results obtained in animal models, the clinical use of Fas ligand (FasL) as an anticancer drug is limited by severe toxicity. Systemic toxicity of death ligands may be prevented by using genes encoding membrane-bound death ligands and by targeted transgene expression through either targeted transduction or targeted transcription. Selective induction of tumor cell death is a promising anticancer strategy. A fusion protein is created by fusing the extracellular domain of Fas ligand (FasL) to the peptide arginine-glycine-aspartic acid (RGD) that selectively targets avβ3-integrins on tumor endothelial cells. The purpose of this study is to evaluate the effects of RGD-FasL on tumor growth and survival in a murine hepatocellular carcinoma (HCC) tumor model. Treatment with RGD-FasL displaying an obvious suppressive effect on the HCC tumor model as compared to that with FasL (p < 0.05) and resulted in a more additive effect on tumor growth delay in this model. RGD-FasL treatment significantly enhanced mouse survival and caused no toxic effect, such as weight loss, organ failure, or other treatment-related toxicities. Apoptosis was detected by flow cytometric analysis and TUNEL assays; those results also showed that RGD-FasL is a more potent inducer of cell apoptosis for H22 and H9101 cell lines than FasL (p < 0.05). In conclusion, RGD-FasL appears to be a low-toxicity selective inducer of tumor cell death, which merits further investigation in preclinical and clinical studies. Furthermore, this approach offers a versatile technology for complexing target ligands with therapeutic recombinant proteins. To distinguish the anti-tumor effects of FasL in vivo, tumor and liver tissues were harvested to examine for evidence of necrotic cells, tumor cells, or apoptotic cells by Hematoxylin and eosin (H&E) staining. PMID:19728930

  11. RGD-conjugated albumin nanoparticles as a novel delivery vehicle in pancreatic cancer therapy.

    PubMed

    Ji, Shunrong; Xu, Jin; Zhang, Bo; Yao, Wantong; Xu, Wenyan; Wu, Wenzhe; Xu, Yongfeng; Wang, Hao; Ni, Quanxing; Hou, Huimin; Yu, Xianjun

    2012-02-15

    Integrin αvβ3 receptor is expressed on several types of cancer cells, including pancreatic cancer cells, and plays an important role in tumor growth and metastasis. The ability to target the integrin αvβ3 receptor on cancer cells increases the efficacy of targeted therapy and reduces the side effects. The aim of this study is to develop a novel arginine-glycine-aspartic acid (RGD) peptide -conjugated albumin nanoparticle to enhance the intracellular uptake of anticancer drug into the pancreatic cancer cells through receptor-mediated endocytosis. In the cellular uptake studies, the fluorescent signal of RGD-conjugated BSANPs in BxPC3 cells was higher than that of BSANPs without RGD conjugation as determined by fluorescence spectrophotometer. We also found that BSANPs bound to BxPC3 cells in a time- and concentration-dependent manner. The uptake of RGD-conjugated BSANPs by pancreatic cancer cells was inhibited by an excess amount of free RGD peptide, indicating that the binding and/or uptake were mediated by the αvβ3 receptor. Furthermore, the nanoparticles were found to be located close to the nuclei by using laser scanning confocal microscopy. Besides, no significant in vitro cytotoxicity was observed as measured with MTT assay. Both in vitro and in vivo antitumor efficacy was improved by targeting gemcitabine-loaded nanoparticles to BxPC-3 cells using RGD peptides. Therefore, the RGD-conjugated BSANPs hold great potential as an effective drug delivery system to deliver therapeutic agents to pancreatic cancer.

  12. RGD-functionalized ultrasmall iron oxide nanoparticles for targeted T1-weighted MR imaging of gliomas

    NASA Astrophysics Data System (ADS)

    Luo, Yu; Yang, Jia; Yan, Yu; Li, Jingchao; Shen, Mingwu; Zhang, Guixiang; Mignani, Serge; Shi, Xiangyang

    2015-08-01

    We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration range, and display targeting specificity to glioma cells overexpressing αvβ3 integrin in vitro. With the relatively high r1 relaxivity (r1 = 1.4 mM-1 s-1), the Fe3O4-PEG-RGD particles can be used as an efficient nanoprobe for targeted T1-weighted positive MR imaging of glioma cells in vitro and the xenografted tumor model in vivo via an active RGD-mediated targeting pathway. The developed RGD-functionalized Fe3O4 NPs may hold great promise to be used as a nanoprobe for targeted T1-weighted MR imaging of different αvβ3 integrin-overexpressing cancer cells or biological systems.We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration

  13. Cellular recognition of synthetic peptide amphiphiles in supported bioartificial membranes

    NASA Astrophysics Data System (ADS)

    Pakalns, Teika

    The goal of this study was to demonstrate that lipidated cell adhesion peptides could form well-ordered biomimetic surfaces that were capable of influencing cellular behavior in a controlled and specific manner. The first step taken was to covalently link synthetic dialkyl tails to the amino-termini of the collagen-derived peptide IV-H1 (amino acid sequence GVKGDKGNPGWPGAP) and the well-known tripeptide Arg-Gly-Asp (RGD) to produce amino-coupled peptide amphiphiles. Other spatial orientations of RGD were also generated by coupling tails to the carboxyl-terminus to give carboxyl-coupled RGD amphiphiles and to both the amino- and carboxyl-termini to give looped RGD amphiphiles. The next step taken was to let the peptide amphiphile self-assemble along with methyl ester-capped dialkyl tails into mixed films. It was found that all the peptide amphiphiles formed stable monolayers at the air-water interface in a Langmuir trough. IV-H1 amphiphiles and carboxyl-coupled and looped RGD amphiphiles deposited well as Langmuir-Blodgett mixed films on solid surfaces at all peptide concentrations, but aminocoupled RGD amphiphiles did not deposit well at high RGD concentrations. FT-IR studies of films containing RGD amphiphiles showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. The final step was to study cellular response to mixed films containing IV-H1 or RGD amphiphiles. The spreading of melanoma cells was influenced by both the molar concentration and spatial orientation of the amphiphilic peptides. Cells spread on IV-H1 and looped RGD films in a concentration-dependent manner, but spread indiscriminately on carboxyl-coupled RGD films and did not spread at all on well-deposited amino-coupled RGD films. The specificity of the cellular response to looped RGD amphiphiles was investigated. Control films of looped Arg-Gly-Glu (RGE) amphiphiles inhibited the adhesion and spreading of melanoma and endothelial cells, and antibody inhibition of the

  14. Modeling the Interaction between Integrin-Binding Peptide (RGD) and Rutile Surface: The Effect of Cation Mediation on Asp Adsorption

    SciTech Connect

    Wu, Chunya; Skelton, Adam; Chen, Mingjun; Vlcek, Lukas; Cummings, Peter T

    2012-01-01

    The binding of a negatively charged residue, aspartic acid (Asp) in tripeptide arginine-glycine-aspartic acid, onto a negatively charged hydroxylated rutile (110) surface in aqueous solution, containing divalent (Mg{sup 2+}, Ca{sup 2+}, or Sr{sup 2+}) or monovalent (Na{sup +}, K{sup +}, or Rb{sup +}) cations, was studied by molecular dynamics (MD) simulations. The results indicate that ionic radii and charges will significantly affect the hydration, adsorption geometry, and distance of cations from the rutile surface, thereby regulating the Asp/rutile binding mode. The adsorption strength of monovalent cations on the rutile surface in the order Na{sup +} > K{sup +} > Rb{sup +} shows a 'reverse' lyotropic trend, while the divalent cations on the same surface exhibit a 'regular' lyotropic behavior with decreasing crystallographic radii (the adsorption strength of divalent cations: Sr{sup 2+} > Ca{sup 2+} > Mg{sup 2+}). The Asp side chain in NaCl, KCl, and RbCl solutions remains stably H-bonded to the surface hydroxyls and the inner-sphere adsorbed compensating monovalent cations act as a bridge between the COO{sup -} group and the rutile, helping to 'trap' the negatively charged Asp side chain on the negatively charged surface. In contrast, the mediating divalent cations actively participate in linking the COO{sup -} group to the rutile surface; thus the Asp side chain can remain stably on the rutile (110) surface, even if it is not involved in any hydrogen bonds with the surface hydroxyls. Inner- and outer-sphere geometries are all possible mediation modes for divalent cations in bridging the peptide to the rutile surface.

  15. Repetitive Arg-Gly-Asp peptide as a cell-stimulating agent on electrospun poly(ϵ-caprolactone) scaffold for tissue engineering.

    PubMed

    Chaisri, Pacharaporn; Chingsungnoen, Artit; Siri, Sineenat

    2013-11-01

    Electrospun scaffolds derived from poly(ϵ-caprolactone) (PCL), a well known biodegradable material, have an architecture that is suitable for hosting cells. However, their biomedical applications are restricted because these scaffolds lack the bioactivity necessary to stimulate cell responses. In this work, a repetitive Arg-Gly-Asp (rRGD) peptide was produced as a cell-stimulating agent to provide the PCL scaffold with bioactivity. DNA encoding rRGD was amplified by polymerase chain reaction using overlap primers without a DNA template, and cloned into a protein expression vector to produce a His-tag fusion peptide. In an in vitro cell adhesion assay, the purified rRGD peptide, comprising 30 RGD repeats, promoted a 1.5-fold greater cell adhesion than the commercial tripeptide RGD. The rRGD peptide was immobilized onto an electrospun PCL scaffold that had been pretreated with argon plasma and graft-polymerized with acrylic acid. Fourier transform infrared (FTIR) analysis indicated that covalently linked rRGD peptide was present on the scaffold. The PCL scaffold with immobilized rRGD showed significantly changed hydrophilic properties and an enhanced adhesion and proliferation of mouse fibroblast cells by 2.3- and 2.9-fold, respectively, compared to the PCL scaffold alone. Through its ability to promote cell adhesion and proliferation, the rRGD peptide has great potential as a stimulant for improving the suboptimal cell-matrix interaction of polymeric scaffolds for tissue engineering applications.

  16. In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the αvβ3 integrin in Kaposi"s sarcoma

    NASA Astrophysics Data System (ADS)

    Kwon, Sunkuk; Ke, Shi; Houston, Jessica P.; Wang, Wei; Wu, Qingping; Li, Chun; Sevick Muraca, Eva M.

    2005-04-01

    The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the αvβ3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi"s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi"s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.

  17. Bioabsorbable Bypass Grafts Biofunctionalised with RGD Have Enhanced Biophysical Properties and Endothelialisation Tested In vivo

    PubMed Central

    Antonova, Larisa V.; Seifalian, Alexander M.; Kutikhin, Anton G.; Sevostyanova, Victoria V.; Krivkina, Evgeniya O.; Mironov, Andrey V.; Burago, Andrey Y.; Velikanova, Elena A.; Matveeva, Vera G.; Glushkova, Tatiana V.; Sergeeva, Evgeniya A.; Vasyukov, Georgiy Y.; Kudryavtseva, Yuliya A.; Barbarash, Olga L.; Barbarash, Leonid S.

    2016-01-01

    Small diameter arterial bypass grafts are considered as unmet clinical need since the current grafts have poor patency of 25% within 5 years. We have developed a 3D scaffold manufactured from natural and synthetic biodegradable polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(𝜀-caprolactone) (PCL), respectively. Further to improve the biophysical properties as well as endothelialisation, the grafts were covalently conjugated with arginine-glycine-aspartic acid (RGD) bioactive peptides. The biophysical properties as well as endothelialisation of PHBV/PCL and PCL 2 mm diameter bypass grafts were assessed with and without biofunctionalisation with RGD peptides in vitro and in vivo. Morphology of the grafts was assessed by scanning electron microscopy, whereas physico-mechanical properties were evaluated using a physiological circulating system equipped with a state of art ultrasound vascular wall tracking system. Endothelialisation of the grafts in vitro and in vivo were assessed using a cell viability assay and rat abdominal aorta replacement model, respectively. The biofunctionalisation with RGD bioactive peptides decreased mean fiber diameter and mean pore area in PHBV/PCL grafts; however, this was not the case for PCL grafts. Both PHBV/PCL and PCL grafts with RGD peptides had lower durability compared to those without; these durability values were similar to those of internal mammary artery. Modification of PHBV/PCL and PCL grafts with RGD peptides increased endothelial cell viability in vitro by a factor of eight and enhanced the formation of an endothelial cell monolayer in vivo 1 month postimplantation. In conclusion, PHBV/PCL small-caliber graft can be a suitable 3D scaffold for the development of a tissue engineering arterial bypass graft. PMID:27252652

  18. 18F-FP-PEG2-β-Glu-RGD2: A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging

    PubMed Central

    Tang, Ganghua; Yao, Shaobo; Yao, Baoguo; Wang, Hongliang; Nie, Dahong; Liang, Xiang; Tang, Caihua; He, Shanzhen

    2015-01-01

    Radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive determination of integrin αvβ3 expression in tumors. In this study, we performed radiosynthesis and biological evaluation of a new 18F-labeled RGD homodimeric peptide with one 8-amino-3,6-dioxaoctanoic acid (PEG2) linker on the glutamate β-amino group (18F-FP-PEG2-β-Glu-RGD2) as a symmetric PET tracer for tumor imaging. Biodistribution studies showed that radioactivity of 18F-FP-PEG2-β-Glu-RGD2 was rapidly cleared from blood by predominately renal excretion. MicroPET-CT imaging with 18F-FP-PEG2-β-Glu-RGD2 revealed high tumor contrast and low background in A549 human lung adenocarcinoma-bearing mouse models, PC-3 prostate cancer-bearing mouse models, and orthotopic transplanted C6 brain glioma models. 18F-FP-PEG2-β-Glu-RGD2 exhibited good stability in vitro and in vivo. The results suggest that this tracer is a potential PET tracer for tumor imaging. PMID:26397833

  19. Peptide interfacial biomaterials improve endothelial cell adhesion and spreading on synthetic polyglycolic acid materials.

    PubMed

    Huang, Xin; Zauscher, Stefan; Klitzman, Bruce; Truskey, George A; Reichert, William M; Kenan, Daniel J; Grinstaff, Mark W

    2010-06-01

    Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed "interfacial biomaterials" (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K (A) 1 x 10(7) M(-1)) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70-120% increase in cell spreading from 30 to 60 minutes after plating.

  20. A free cysteine prolongs the half-life of a homing peptide and improves its tumor-penetrating activity

    PubMed Central

    Pang, Hong-Bo; Braun, Gary B.; She, Zhi-Gang; Kotamraju, Venkata R.; Sugahara, Kazuki N.; Teesalu, Tambet; Ruoslahti, Erkki

    2014-01-01

    The accessibility of extravascular tumor tissue to drugs is critical for therapeutic efficacy. We previously described a tumor-targeting peptide (iRGD) that elicits active transport of drugs and macromolecules (covalently coupled or co-administered) across the vascular wall into tumor tissue. Short peptides (iRGD is a 9-amino acid cyclic peptide) generally have a plasma half-life measured in minutes. Since short half-life limits the window of activity obtained with a bolus injection of iRGD, we explored to extend the half-life of the peptide. We show here that addition of a cysteine residue prolongs the plasma half-life of iRGD and increases the accumulation of the peptide in tumors. This modification prolongs the activity of iRGD in inducing macromolecular extravasation and leads to greater drug accumulation in tumors than is obtained with the unmodified peptide. This effect is mediated by covalent binding of iRGD to plasma albumin through a disulfide bond. Our study provides a simple strategy to improve peptide pharmacokinetics and activity. Applied to RGD, it provides a means to increase the entry of therapeutic agents into tumors. PMID:24345789

  1. Cyclic RGD-poly(ethylene glycol)-polyethyleneimine is more suitable for glioblastoma targeting gene transfer in vivo.

    PubMed

    Zhan, Changyou; Qian, Jun; Feng, Linglin; Zhong, Gaoren; Zhu, Jianhua; Lu, Weiyue

    2011-08-01

    Arginine-glycine-aspartic acid (RGD) is a widely chosen ligand to improve the specific gene targeting transfection efficiency of polyethyleneimine (PEI) in vivo. However, the optimal RGD conjugating mode, RGD-poly(ethylene glycol)-PEI (RGD-PEG-PEI) or RGD-PEI-methoxyl poly(ethylene glycol) (RGD-PEI-mPEG) still remains controversial. In this study, RGD-PEG-PEI and RGD-PEI-mPEG were synthesized and compared with respects to their glioblastoma cell-binding capability and tumor-targeting ability of their complexes with plasmid DNA. These results demonstrated that RGD-PEG-PEI/plasmid enhanced green fluorescent protein (pEGFP)-N2 complexes had higher binding affinities with U87 cells than RGD-PEI-mPEG/pEGFP-N2 complexes. The gene transfection was also performed on U87 cells in vitro and in vivo. In vitro, both of the RGD-modified PEI derivatives enhanced the gene transfection efficiency to some extent. However, all of the complexes (with or without RGD modification) had high transfection efficiency. The biodistribution of RGD-PEG-PEI/pEGFP-N2 complexes in mice bearing subcutaneous glioblastomas were significantly greater than that of RGD-PEI-mPEG/pEGFP-N2 complexes, suggesting a more efficient gene transfection in vivo. In the RGD-PEG-PEI, the use of a PEG spacer was particularly important. These results indicated that RGD-PEG-PEI was more suitable for targeted gene transfer in vivo.

  2. Targeted systemic delivery of siRNA to cervical cancer model using cyclic RGD-installed unimer polyion complex-assembled gold nanoparticles.

    PubMed

    Yi, Yu; Kim, Hyun Jin; Mi, Peng; Zheng, Meng; Takemoto, Hiroyasu; Toh, Kazuko; Kim, Beob Soo; Hayashi, Kotaro; Naito, Mitsuru; Matsumoto, Yu; Miyata, Kanjiro; Kataoka, Kazunori

    2016-12-28

    For systemic delivery of small interfering RNA (siRNA) to solid tumors, we developed an actively-targeted unimer polyion complex-assembled gold nanoparticle (uPIC-AuNP) by a two-step assembling process. First is the monodispersed uPIC formation from the single molecules of therapeutic siRNA and the block catiomer, cyclic RGD (cRGD) peptide-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (cRGD-PEG-PLL-LA). Second is the surface decoration of a 20nm-sized AuNP with uPICs. The cRGD-installed uPIC-AuNPs (cRGD-uPIC-AuNP) provided the targetability for selective binding to the cancer and cancer-related endothelial cellular surface, while regulating their size <50nm with a quite narrow distribution. The targeting efficacy of the cRGD-uPIC-AuNP was confirmed by in vitro cellular uptake in cultured cervical cancer (HeLa) cells and in vivo tumor accumulation in a subcutaneous HeLa model after systemic administration, compared with a non-targeted control uPIC-AuNP. Due to the targetability of the ligand, the cRGD-uPIC-AuNP achieved the significantly enhanced gene silencing ability in the subcutaneous HeLa tumor. Ultimately, the systemic delivery of siRNA targeted for papilloma virus-derived E6 oncogene by cRGD-uPIC-AuNP significantly inhibited the growth of subcutaneous HeLa tumor. This research demonstrates that the bottom-up construction of nanocarriers using monodispersed building blocks can be employed as delivery platforms for RNA interference-based cancer therapy.

  3. Rhenium and technetium tricarbonyl complexes of 1,4-Substituted pyridyl-1,2,3-triazole bidentate 'click' ligands conjugated to a targeting RGD peptide.

    PubMed

    Connell, Timothy U; Hayne, David J; Ackermann, Uwe; Tochon-Danguy, Henri J; White, Jonathan M; Donnelly, Paul S

    2014-04-01

    New 1,4-substituted pyridyl-1,2,3-triazole ligands with pendent phenyl isothiocyanate functional groups linked to the heterocycle through a short methylene or longer polyethylene glycol spacers were prepared and conjugated to a peptide containing the arginine-glycine-aspartic acid peptide motif. Rhenium and technetium carbonyl complexes, [M(CO)3 L(x) (py)](+) (where M = Re(I) or (99m) Tc(I) ; L(x)  = 1,4-substituted pyridyl-1,2,3-triazole ligands and py = pyridine) were prepared. One rhenium complex has been characterized by X-ray crystallography, and the luminescent properties of [M(CO)3 L(x) (py)](+) are reported.

  4. Arginine-glycine-aspartic acid- and fibrinogen gamma-chain carboxyterminal peptides inhibit platelet adherence to arterial subendothelium at high wall shear rates. An effect dissociable from interference with adhesive protein binding.

    PubMed Central

    Lawrence, J B; Kramer, W S; McKeown, L P; Williams, S B; Gralnick, H R

    1990-01-01

    Arg-Gly-Asp (RGD)- and fibrinogen gamma-chain carboxyterminal (GQQHHLGGAKQAGDV) peptides inhibit fibrinogen, fibronectin (Fn), vitronectin, and von Willebrand factor (vWF) binding to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). GP IIb-IIIa, vWF, and Fn are essential for normal platelet adherence to subendothelium. We added peptides to normal citrated whole blood before perfusion over human umbilical artery subendothelium and evaluated platelet adherence morphometrically at high (2,600 s-1) and low (800 s-1) wall shear rates. We also examined the effects of the peptides on platelet adhesion to collagen in a static system. At the high wall shear rate, RGDS and GQQHHLGGAKQAGDV caused dose-dependent reduction in the surface coverage with spread and adherent platelets. Amino acid transposition and conservative substitutions of RGD peptides and the AGDV peptide significantly inhibited platelet adherence at 2,600 s-1. By contrast, the modified RGD peptides and AGDV do not affect adhesive protein binding to platelets. None of the native or modified RGD- or fibrinogen gamma-chain peptides significantly inhibited either platelet adherence to subendothelium at 800 s-1 or platelet adhesion to collagen. Our findings demonstrate that peptides that interfere with adhesive protein binding to GP IIb-IIIa inhibit platelet adherence to vascular subendothelium with flowing blood only at high wall shear rates. Platelet adherence to subendothelium at high wall shear rates appears to be mediated by different recognition specificities from those required for fluid-phase adhesive protein binding or static platelet adhesion. PMID:2243140

  5. rLj-RGD3, a Novel Recombinant Toxin Protein from Lampetra japonica, Protects against Cerebral Reperfusion Injury Following Middle Cerebral Artery Occlusion Involving the Integrin-PI3K/Akt Pathway in Rats

    PubMed Central

    Jiang, Junshu; Wang, Shengnan; Jia, Qilan; Wang, Yue; Li, Weiping; Zhou, Qin; Lv, Li; Li, Qingwei

    2016-01-01

    Background The RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the Lampetra japonica fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model. We also explored the mechanism by which rLj-RGD3 acts by measuring protein and mRNA expression levels, with an emphasis on the FAK and integrin-PI3K/Akt anti-apoptosis pathways. Methods rLj-RGD3 was obtained from the buccal secretions of Lampetra japonica using gene recombination technology. Sprague Dawley (SD) rats were randomly divided into the following seven groups: a sham group; a vehicle-treated (VT) group; 100.0 μg·kg-1, 50.0 μg·kg-1 and 25.0 μg·kg-1 dose rLj-RGD3 groups; and two positive controls, including 1.5 mg·kg-1 Edaravone (ED) and 100.0 μg·kg-1 Eptifibatide (EP). MCAO was induced using a model consisting of 2 h of ischemia and 24 h of reperfusion. Behavioral changes were observed in the normal and operation groups after focal cerebral ischemia/reperfusion was applied. In addition, behavioral scores were evaluated at 4 and 24 h after reperfusion. Brain infarct volumes were determined based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining. Moreover, neuronal apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. We determined the expression levels of focal adhesion kinase (FAK), phosphatidyl inositol 3-kinase (PI3K

  6. Immobilization of cell adhesive RGD peptide onto the surface of highly porous biodegradable polymer scaffolds fabricated by a gas foaming/salt leaching method.

    PubMed

    Yoon, Jun Jin; Song, Soon Ho; Lee, Doo Sung; Park, Tae Gwan

    2004-11-01

    A cell adhesive peptide moiety, Gly-Arg-Gly-Asp-Tyr (GRGDY), was immobilized onto the surface of highly porous biodegradable polymer scaffolds for enhancing cell adhesion and function. A carboxyl terminal end of poly(D,L-lactic-co-glycolic acid) (PLGA) was functionalized with a primary amine group by conjugating hexaethylene glycol-diamine. The PLGA-NH2 was blended with PLGA in varying ratios to prepare films by solvent casting or to fabricate porous scaffolds by a gas foaming/salt leaching method. Under hydrating conditions, the activated GRGDY could be directly immobilized to the surface exposed amine groups of the PLGA-NH2 blend films or scaffolds. For the PLGA blend films, the surface density of GRGDY, surface wettability change, and cell adhesion behaviors were characterized. The extent of cell adhesion was substantially enhanced by increasing the blend ratio of PLGA-NH2 to PLGA. The level of an alkaline phosphatase activity, measured as a degree of cell differentiation, was also enhanced as a result of the introduction of cell adhesive peptides.

  7. RGD-Xyloside Conjugates Prime Glycosaminoglycans

    PubMed Central

    Tran, Vy M.; Victor, Xylophone V.; Yockman, James W.; Kuberan, Balagurunathan

    2010-01-01

    Glycosaminoglycans (GAG) play decisive roles in various cardio-vascular & cancer-associated processes. Changes in the expression of GAG fine structures, attributed to deregulation of their biosynthetic and catabolic enzymes, are hallmarks of vascular dysfunction and tumor progression. Wide spread role of GAG chains in blood clotting, wound healing and tumor biology has led to the development of modified GAG chains, GAG binding peptides and GAG based enzyme inhibitors as therapeutic agents. Xylosides, carrying hydrophobic aglycone, are known to induce GAG biosynthesis in various systems. Given the important roles of GAG chains in vascular and tumor biology, we envision that RGD-conjugated xylosides could be targeted to activated endothelial and cancer cells, which are known to express αvβ3 integrin, and thereby, modulate the pathological processes. To accomplish this vision, xylose residue was conjugated to linear and cyclic RGD containing peptides using click chemistry. Our results demonstrate that RGD-conjugated xylosides are able to prime GAG chains in various cell types, and future studies are aimed toward evaluating potential utility of such xylosides in treating myocardial infarction as well as cancer-associated thrombotic complications. PMID:20717719

  8. Multilayered polyion complexes with dissolvable silica layer covered by controlling densities of cRGD-conjugated PEG chains for cancer-targeted siRNA delivery.

    PubMed

    Naito, Mitsuru; Azuma, Ryota; Takemoto, Hiroyasu; Hori, Mao; Yoshinaga, Naoto; Osawa, Shigehito; Kamegawa, Rimpei; Kim, Hyun Jin; Ishii, Takehiko; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

    2017-03-12

    Surface functionalization of nanoparticles is a crucial factor for nanoparticle-mediated drug and nucleic acid delivery. Particularly, the density of targeting ligands on nanoparticle significantly affects the affinity of nanoparticles to specific cellular surface (or receptor) through the multivalent binding effect. Herein, multilayered polyion complexes (mPICs) are prepared to possess varying densities of cyclic RGD peptide (cRGD) ligands for cancer-targeted small interfering RNA (siRNA) delivery. A template PIC is first prepared by mixing siRNAs with homo catiomers of N-substituted polyaspartamide bearing tetraethylenepentamine (PAsp(TEP)) in aqueous solution, followed by silica-coating through silicate condensation reaction. Then, silica-coated PICs (sPICs) are further covered with block catiomers of PAsp(TEP) and poly(ethylene glycol) (PEG) equipped with cRGD ligand. Successful preparation of targeted mPICs is confirmed from the changes in size and ζ-potential and the elemental analysis by transmission electron microscopy. Notably, the number of cRGD ligands per mPIC is regulated by altering the silicate concentration upon preparation of sPICs, which is confirmed by fluorescence correlation spectroscopy using fluorescent-labeled block catiomers. Ultimately, the targeted mPICs with a higher number of cRGD ligands demonstrate more efficient cellular uptake in cultured cancer cells, leading to enhanced gene silencing activity.

  9. Nanotechnology for delivery of peptide nucleic acids (PNAs).

    PubMed

    Gupta, Anisha; Bahal, Raman; Gupta, Meera; Glazer, Peter M; Saltzman, W Mark

    2016-10-28

    Over the past three decades, peptide nucleic acids have been employed in numerous chemical and biological applications. Peptide nucleic acids possess enormous potential because of their superior biophysical properties, compared to other oligonucleotide chemistries. However, for therapeutic applications, intracellular delivery of peptide nucleic acids remains a challenge. In this review, we summarize the progress that has been made in delivering peptide nucleic acids to intracellular targets. In addition, we emphasize recent nanoparticle-based strategies for efficient delivery of conventional and chemically-modified peptides nucleic acids.

  10. Gold Nanorods Conjugated with Doxorubicin and cRGD for Combined Anticancer Drug Delivery and PET Imaging

    PubMed Central

    Xiao, Yuling; Hong, Hao; Matson, Vyara Z.; Javadi, Alireza; Xu, Wenjin; Yang, Yunan; Zhang, Yin; Engle, Jonathan W.; Nickles, Robert J.; Cai, Weibo; Steeber, Douglas A.; Gong, Shaoqin

    2012-01-01

    A multifunctional gold nanorod (GNR)-based nanoplatform for targeted anticancer drug delivery and positron emission tomography (PET) imaging of tumors was developed and characterized. An anti-cancer drug (i.e., doxorubicin (DOX)) was covalently conjugated onto PEGylated (PEG: polyethylene glycol) GNR nanocarriers via a hydrazone bond to achieve pH-sensitive controlled drug release. Tumor-targeting ligands (i.e., the cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides, cRGD) and 64Cu-chelators (i.e., 1,4,7-triazacyclononane-N, N', N''-triacetic acid (NOTA)) were conjugated onto the distal ends of the PEG arms to achieve active tumor-targeting and PET imaging, respectively. Based on flow cytometry analysis, cRGD-conjugated nanocarriers (i.e., GNR-DOX-cRGD) exhibited a higher cellular uptake and cytotoxicity than non-targeted ones (i.e., GNR-DOX) in vitro. However, GNR-DOX-cRGD and GNR-DOX nanocarriers had similar in vivo biodistribution according to in vivo PET imaging and biodistribution studies. Due to the unique optical properties of GNRs, this multifunctional GNR-based nanoplatform can potentially be optimized for combined cancer therapies (chemotherapy and photothermal therapy) and multimodality imaging (PET, optical, X-ray computed tomography (CT), etc.). PMID:22916075

  11. [Status and advances of RGD molecular imaging in lung cancer].

    PubMed

    Yue, Ning; Yuan, Shuanghu; Yang, Guoren

    2014-12-01

    Lung cancer has been one of the most common and the highest mortality rates malignant tumors at home and abroad. Sustained angiogenesis was not only the characteristic of malignant tumors, but also the foundation of tumor proliferation, invasion, recurrence and metastasis, it was also one of the hot spots of treatments in lung cancer biology currently. Integrins played an important part in tumor angiogenesis. Arg-Gly-Asp (RGD) peptides could combine with integrins specifically, and the application of radionuclide-labeled RGD molecular probes enabled imaging of tumor blood vessels to reflect its changes. The lung cancer imaging of RGD peptides at home and abroad in recent years was reviewed in this article.

  12. F-18 Labeled RGD Probes Based on Bioorthogonal Strain-Promoted Click Reaction for PET Imaging.

    PubMed

    Kim, Hye Lan; Sachin, Kalme; Jeong, Hyeon Jin; Choi, Wonsil; Lee, Hyun Soo; Kim, Dong Wook

    2015-04-09

    A series of fluorine-substituted monomeric and dimeric cRGD peptide derivatives, such as cRGD-ADIBOT-F (ADIBOT = azadibenzocyclooctatriazole), di-cRGD-ADIBOT-F, cRGD-PEG5-ADIBOT-F, and di-cRGD-PEG5-ADIBOT-F, were prepared by strain-promoted alkyne azide cycloaddition (SPAAC) reaction of the corresponding aza-dibenzocyclooctyne (ADIBO) substituted peptides with a fluorinated azide 3. Among these cRGD derivatives, di-cRGD-PEG5-ADIBOT-F had the highest binding affinity in a competitive binding assay compared to other derivatives and even the original cRGDyk. On the basis of the in vitro study results, di-cRGD-PEG5-ADIBOT-(18)F was prepared from a SPAAC reaction with (18)F-labeled azide and subsequent chemo-orthogonal scavenger-assisted separation without high performance liquid chromatography (HPLC) purification in 92% decay-corrected radiochemical yield (dcRCY) with high specific activity for further in vivo positron emission tomography (PET) imaging study. In vivo PET imaging study and biodistribution data showed that this radiotracer allowed successful visualization of tumors with good tumor-to-background contrast and significantly higher tumor uptake compared to other major organs.

  13. Bone healing of commercial oral implants with RGD immobilization through electrodeposited poly(ethylene glycol) in rabbit cancellous bone.

    PubMed

    Park, Jin-Woo; Kurashima, Kazuya; Tustusmi, Yusuke; An, Chang-Hyeon; Suh, Jo-Young; Doi, Hisashi; Nomura, Naoyuki; Noda, Kazuhiko; Hanawa, Takao

    2011-08-01

    Immobilization of RGD peptides on titanium (Ti) surfaces enhances implant bone healing by promoting early osteoblastic cell attachment and subsequent differentiation by facilitating integrin binding. Our previous studies have demonstrated the efficacy of RGD peptide immobilization on Ti surfaces through the electrodeposition of poly(ethylene glycol) (PEG) (RGD/PEG/Ti), which exhibited good chemical stability and bonding. The RGD/PEG/Ti surface promoted differentiation and mineralization of pre-osteoblasts. This study investigated the in vivo bone healing capacity of the RGD/PEG/Ti surface for biomedical application as a more osteoconductive implant surface in dentistry. The RGD/PEG/Ti surface was produced on an osteoconductive implant surface, i.e. the grit blasted micro-rough surface of a commercial oral implant. The osteoconductivity of the RGD/PEG/Ti surface was compared by histomorphometric evaluation with an RGD peptide-coated surface obtained by simple adsorption in rabbit cancellous bone after 2 and 4 weeks healing. The RGD/PEG/Ti implants displayed a high degree of direct bone apposition in cancellous bone and achieved greater active bone apposition, even in areas of poor surrounding bone. Significant increases in the bone to implant contact percentage were observed for RGD/PEG/Ti implants compared with RGD-coated Ti implants obtained by simple adsorption both after 2 and 4 weeks healing (P<0.05). These results demonstrate that RGD peptide immobilization on a Ti surface through electrodeposited PEG may be an effective method for enhancing bone healing with commercial micro-rough surface oral implants in cancellous bone by achieving rapid bone apposition on the implant surface.

  14. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    PubMed

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  15. Fmoc/Trt-amino acids: comparison to Fmoc/tBu-amino acids in peptide synthesis.

    PubMed

    Barlos, K; Gatos, D; Koutsogianni, S

    1998-03-01

    Model peptides containing the nucleophilic amino acids Trp and Met have been synthesized with the application of Fmoc/Trt- and Fmoc/tBu-amino acids, for comparison. The deprotection of the peptides synthesized using Fmoc/Trt-amino acids in all cases leads to crude peptides of higher purity than that of the same peptides synthesized using Fmoc/tBu-amino acids.

  16. Radiolabeling of a cyclic RGD (cyclo Arg-Gly-Asp-d-Tyr-Lys) peptide using sodium hypochlorite as an oxidizing agent.

    PubMed

    Doll, Stephanie; Woolum, Karen; Kumar, Krishan

    2016-09-01

    A simple and rapid nonradioactive iodide labeling/radiolabeling method for peptides, using an inexpensive oxidizing agent such as sodium hypochlorite and a cyclic peptide, cRGDyK (cyclo Arg-Gly-Asp-d-Tyr-Lys), was developed in this work. Labeling reaction was optimized by conducting experiments under variable ratios of the reagents, the reaction times, and the pH. The study demonstrated that radiolabeling of the cyclic peptide was fast and pH independent. Monoiodinated and di-iodinated cRGDyK were formed under all conditions and varied with the ratio of the reagents and the reaction time. Total percent of the iodinated cRGDyK (monoiodinated and di-iodinated cRGDyK) varied between 44 and 100 depending on the reaction conditions. Excess cyclic peptide over equal molar ratio of sodium iodide and sodium hypochlorite yielded in predominant amounts of monoiodinated cRGDyK, ie, >60% under 2:1:1 ratio and ~88% under 5:1:1 ratio of cRGDyK:sodium iodide:sodium hypochlorite.

  17. The effect of conjugating RGD into 3D alginate hydrogels on adipogenic differentiation of human adipose-derived stromal cells.

    PubMed

    Kang, Sun-Woong; Cha, Byung-Hyun; Park, Honghyun; Park, Kwang-Sook; Lee, Kuen Yong; Lee, Soo-Hong

    2011-05-12

    The effects of RGD peptide conjugation to alginate hydrogel on the adipogenic differentiation of ASCs was investigated. After 3 d of culture, RGD-modified alginate hydrogels significantly stimulated FAK and integrin α1 gene expressions and vinculin expression in ASCs. In addition, RGD-modified alginate hydrogels significantly enhanced the adipogenic differentiation of human ASCs to exhibit higher expression levels of oil red O staining and adipogenic genes compared to those of the control group (unmodified gels). These results suggest potential applications of RGD-modified alginate gels for adipose tissue regeneration.

  18. RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

    PubMed Central

    Hu, Qiao; Wang, Xiao-Yan; Kang, Li-Ke; Wei, Hai-Ming; Xu, Chun-Mei; Wang, Tao; Wen, Zong-Hua

    2016-01-01

    Objective To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis. Methods RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression. Results The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01). Conclusions RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts. PMID:26862757

  19. A stapled peptide antagonist of MDM2 carried by polymeric micelles sensitizes glioblastoma to temozolomide treatment through p53 activation.

    PubMed

    Chen, Xishan; Tai, Lingyu; Gao, Jie; Qian, Jianchang; Zhang, Mingfei; Li, Beibei; Xie, Cao; Lu, Linwei; Lu, Wuyuan; Lu, Weiyue

    2015-11-28

    Antagonizing MDM2 and MDMX to activate the tumor suppressor protein p53 is an attractive therapeutic paradigm for the treatment of glioblastoma multiforme (GBM). However, challenges remain with respect to the poor ability of p53 activators to efficiently cross the blood-brain barrier and/or blood-brain tumor barrier and to specifically target tumor cells. To circumvent these problems, we developed a cyclic RGD peptide-conjugated poly(ethylene glycol)-co-poly(lactic acid) polymeric micelle (RGD-M) that carried a stapled peptide antagonist of both MDM2 and MDMX (sPMI). The peptide-carrying micelle RGD-M/sPMI was prepared via film-hydration method with high encapsulation efficiency and loading capacity as well as ideal size distribution. Micelle encapsulation dramatically increased the solubility of sPMI, thus alleviating its serum sequestration. In vitro studies showed that RGD-M/sPMI efficiently inhibited the proliferation of glioma cells in the presence of serum by activating the p53 signaling pathway. Further, RGD-M/sPMI exerted potent tumor growth inhibitory activity against human glioblastoma in nude mouse xenograft models. Importantly, the combination of RGD-M/sPMI and temozolomide--a standard chemotherapy drug for GBM increased antitumor efficacy against glioblastoma in experimental animals. Our results validate a combination therapy using p53 activators with temozolomide as a more effective treatment for GBM.

  20. Peptide and amino acid separation with nanofiltration membranes

    SciTech Connect

    Tsuru, Toshinori; Shutou, Takatoshi; Nakao, Shin-Ichi; Kimura, Shoji )

    1994-05-01

    Several nanofiltration membranes [UTC-20, 60 (Toray Industries), NF-40 (Film-Tech Corporation), Desal-5, G-20 (Desalination Systems), and NTR-7450 (Nitto Electric Industrial Co.)] were applied to separate amino acids and peptides on the basis of charge interaction with the membranes since most of them contain charged functional groups. Nanofiltration membranes having a molecular weight cutoff (MWCO) below 300 (UTC-20, 60, NF-40 and Desal-5) were not suitable for separation of amino acids. On the other hand, separation of amino acids and peptides with nanofiltration membranes having a MWCO around 2000-3000 (NTR-7450 and G-20) was satisfactory based on a charge effect mechanism; charged amino acids and peptides were rejected while neutral amino acids and peptides permeated through the membranes. Separation of peptides having different isoelectric points with nanofiltration membranes was possible by adjusting the pH. 15 refs., 11 figs., 4 tabs.

  1. BIOACTIVE PROTEINS, PEPTIDES, AND AMINO ACIDS FROM MACROALGAE(1).

    PubMed

    Harnedy, Pádraigín A; FitzGerald, Richard J

    2011-04-01

    Macroalgae are a diverse group of marine organisms that have developed complex and unique metabolic pathways to ensure survival in highly competitive marine environments. As a result, these organisms have been targeted for mining of natural biologically active components. The exploration of marine organisms has revealed numerous bioactive compounds that are proteinaceous in nature. These include proteins, linear peptides, cyclic peptides and depsipeptides, peptide derivatives, amino acids, and amino acid-like components. Furthermore, some species of macroalgae have been shown to contain significant levels of protein. While some protein-derived bioactive peptides have been characterized from macroalgae, macroalgal proteins currently still represent good candidate raw materials for biofunctional peptide mining. This review will provide an overview of the important bioactive amino-acid-containing compounds that have been identified in macroalgae. Moreover, the potential of macroalgal proteins as substrates for the generation of biofunctional peptides for utilization as functional foods to provide specific health benefits will be discussed.

  2. Biomimetic Hydrogels Incorporating Polymeric Cell-Adhesive Peptide to Promote the 3D Assembly of Tumoroids

    PubMed Central

    Hao, Ying; Zerdoum, Aidan B.; Stuffer, Alexander J.; Rajasekaran, Ayyappan K.; Jia, Xinqiao

    2016-01-01

    Towards the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ~95 μm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD and displayed cortical organization of F-actin. Compared to the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD is a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs. PMID:27723964

  3. [Expression and bioactivity effects to Hela of recombinant toxin protein rLj-RGD3 from Lampetra japonica].

    PubMed

    Zhang, Piqiao; Wang, Jihong; Liu, Xin; Chu, Dan; Li, Qingwei

    2009-05-01

    Lj-RGD3 was a toxin from the saliva gland of Lampetra japonica. To study the anti-tumor function of rLj-RGD3 and confirm its biological status and significance, we extracted total RNA from the saliva gland and amplified the cDNA of Lj-RGD3 by RT-PCR. The cDNA of Lj-RGD3 was 357 bp long and encoded a polypeptide composed of 118 amino acids including 2 cysteines, 17 histidines and 3 RGD (Arg-Gly-Asp) motifs. We cloned the cDNA into the plasmid pET23b, and expressed the recombinant protein rLj-RGD3 in Escherichia coli BL21. Fusion rLj-RGD3 with the C-terminal his-tag was a 15 kD soluble protein. Using the His-Bind affinity chromatography, we purified rLj-RGD3. Furthermore, we determined the biological activities of rLj-RGD3. To examine the ability of rLj-RGD3 inhibiting Hela cells proliferation, we used MTT assay. The results showed that, rLj-RGD3 inhibited bFGF induced proliferation of Hela cells in a dose-dependent manner, the IC50 value was 2.6 micromol/L. Hoechst staining assay revealed that, the nuclei of the cells treated with rLj-RGD3 were stained much brighter than that of untreated cells due to chromatin condensation. Furthermore, the DNA ladder patterns from the cells treated with rLj-RGD3 were also observed. These results demonstrated that rLj-RGD3 could induce apoptosis of Hela cells. Cell adhesion, migration and invasion are critical processes in tumor metastasis. rLj-RGD3 significantly inhibited adhesion of Hela cells to vironectin in a dose-dependent manner. In order to determine the effect of rLj-RGD3 on Hela cells migration toward bFGF, we used Transwell containing insert filter. rLj-RGD3 showed a significant inhibition on Hela cells migration, the inhibition rate was 60%. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD3 significantly inhibited bFGF induced invasion of Hela cells. Taken together, these results revealed that rLj-RGD3 had typical functions

  4. Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain.

    PubMed

    Couto, J R; Taylor, M R; Godwin, S G; Ceriani, R L; Peterson, J A

    1996-04-01

    The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation.

  5. The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.

    PubMed

    Yokosaki, Y; Palmer, E L; Prieto, A L; Crossin, K L; Bourdon, M A; Pytela, R; Sheppard, D

    1994-10-28

    We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.

  6. Fatty acid conjugation enhances the activities of antimicrobial peptides.

    PubMed

    Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

    2013-04-01

    Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

  7. RGD-fatty alcohol-modified docetaxel liposomes improve tumor selectivity in vivo.

    PubMed

    Li, Yinghuan; Zheng, Xuelian; Sun, Yi; Ren, Zhao; Li, Xuemei; Cui, Guohui

    2014-07-01

    The tripeptide arginine-glycine-aspartate (RGD) was conjugated with various fatty alcohols to obtain RGDOCnH2n+1 (n=8, 10, 12, 14, 16, 18), which were incorporated into the bilayer of docetaxel liposomes to improve their tumor specificity. The fatty alcohols were accepted as linking groups to insert the tetrapeptide RGDX (X=amino acid) into the bilayer of liposomes. RGDX was previously shown to be a potent ligand to target tumor cell-surface integrin receptors, whereas RGD was not shown to have this ability. We hypothesized that RGD-fatty alcohol conjugates lacking the fourth amine X can guide liposomes to tumors without reducing their binding affinity to integrins. Antitumor activity, pharmacokinetics and biodistribution studies were evaluated in mice inoculated with S180 sarcoma. Compared with unmodified liposomes, RGD-fatty alcohol-modified liposomes successfully delivered significantly more docetaxel to tumors, which led to significant tumor weight loss and increased tumor docetaxel concentrations accompanied by reduced liver accumulation. Improved affinity of RGD-fatty alcohols to integrins was also confirmed on A375 cell model. Further comparisons among the tumor-targeting capacities of liposomes containing RGD-fatty alcohols, RGDF-fatty alcohols and RGDV-fatty acids demonstrated that RGD-fatty alcohols were as effective as the other two tetrapeptide derivatives. Therefore, a simplified tumor-targeting delivery system using RGD-fatty alcohols was developed.

  8. Differential gene expression of RAW 264.7 macrophages in response to the RGD peptide lunasin with and without lipopolysaccharide stimulation.

    PubMed

    Dia, Vermont P; de Mejia, Elvira Gonzalez

    2011-10-01

    Lunasin is a novel peptide from soybean with demonstrated chemopreventive property. We compared the effect of lunasin on gene expression of RAW 264.7 macrophages with and without lipopolysaccharide (LPS)-stimulation. Our hypothesis was that lunasin will have a differential effect in RAW 264.7 gene expression in a normal and challenged state. Analysis of the microarray data using False Discovery Rate (FDR) method resulted in the identification of 340 up-regulated and 162 down-regulated genes (FDR p-value <0.05) associated with simultaneous treatment of lunasin and LPS for 24h. Treatment of lunasin with no LPS for 24h resulted in the up-regulation of 855 genes and down-regulation of 397 genes. Pre-treatment of lunasin for 24h resulted in the up-regulation of 35 genes and down-regulation of 65 genes in LPS-stimulated RAW 264.7 macrophages. GeneVenn analysis of these three sets of genes showed that there are 66 genes common among the three groups which are mostly associated with regulation of cell death, ion binding and transcription as datamined by DAVID. Analysis of the 838 genes unique to lunasin alone by functional annotation clustering tool showed that lunasin mostly affected genes associated with RNA processing, apoptosis and protein kinase activity. Further datamining of these genes by ingenuity pathway analysis (IPA) showed that lunasin affected genes involved in cellular growth and proliferation, cellular function and maintenance, and cell to cell signaling and interaction. These findings support the potential chemopreventive and chemotherapeutic use of lunasin against cancer.

  9. Amino Acid and Peptide Immobilization on Oxidized Nanocellulose: Spectroscopic Characterization

    PubMed Central

    Barazzouk, Saïd; Daneault, Claude

    2012-01-01

    In this work, oxidized nanocellulose (ONC) was synthesized and chemically coupled with amino acids and peptides using a two step coupling method at room temperature. First, ONC was activated by N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride, forming a stable active ester in the presence of N-hydroxysuccinimide. Second, the active ester was reacted with the amino group of the amino acid or peptide, forming an amide bond between ONC and the grafted molecule. Using this method, the intermolecular interaction of amino acids and peptides was avoided and uniform coupling of these molecules on ONC was achieved. The coupling reaction was very fast in mild conditions and without alteration of the polysaccharide. The coupling products (ONC-amino acids and ONC-peptides) were characterized by transmission electron microscopy and by the absorption, emission, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) spectroscopic techniques.

  10. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  11. Fragmentation reactions of deprotonated peptides containing aspartic acid

    NASA Astrophysics Data System (ADS)

    Harrison, Alex G.; Young, Alex B.

    2006-09-01

    The fragmentation reactions of deprotonated peptides containing aspartic acid have been elucidated using MS2 and MS3 experiments and accurate mass measurements where necessary. The disposition of labile (N and O bonded) hydrogens in the fragmentation products has been studied by exchanging the labile hydrogens for deuterium whereby the [MD]- ion is formed on electrospray ionization. [alpha]-Aspartyl and [beta]-aspartyl dipeptides give very similar fragment ion spectra on collisional activation, involving for both species primarily formation of the y1 ion and loss of H2O from [MH]- followed by further fragmentation, thus precluding the distinction of the isomeric species by negative ion tandem mass spectrometry. Dipeptides of sequence HXxxAspOH give characteristic spectra different from the [alpha]- and [beta]-isomers. For larger peptides containing aspartic acid a common fragmentation reaction involves nominal cleavage of the NC bond N-terminal to the aspartic acid residue to form a c ion (deprotonated amino acid amide (c1) or peptide amide (cn)) and the complimentary product involving elimination of a neutral amino acid amide or peptide amide. When aspartic acid is in the C-terminal position this fragmentation reaction occurs from the [MH]- ion while when the aspartic acid is not in the C-terminal position the fragmentation reaction occurs mainly from the [MHH2O]- ion. The products of this NC bond cleavage reaction serve to identify the position of the aspartic acid residue in the peptide.

  12. Increasing Tumor Accessibility with Conjugatable Disulfide-Bridged Tumor-Penetrating Peptides for Cancer Diagnosis and Treatment

    PubMed Central

    Kotamraju, Venkata Ramana; Sharma, Shweta; Kolhar, Poornima; Agemy, Lilach; Pavlovich, James; Ruoslahti, Erkki

    2015-01-01

    Tumor-homing peptides with tissue-penetrating properties increase the efficacy of targeted cancer therapy by delivering an anticancer agent to the tumor interior. LyP-1 (CGNKRTRGC) and iRGD (CRGDKGPDC) are founding members of this class of peptides. The presence of the cysteines forming the cyclizing disulfide bond complicates conjugation of these peptides with other molecules, such as drugs. Here, we report the synthesis of conjugatable disulfide-bridged peptides and their conjugation to biologically important molecules. We have synthesized the LyP-1, iRGD, and CRGDC (GACRGDCLGA) peptides with a cysteine or maleimidohexanoic acid added externally at N-terminus of the sequences. Subsequent conjugation to payloads yielded stable compounds in which the tumor-homing properties of the peptide and the biological activity of the payload were retained. PMID:27385913

  13. Cellular Density Effect on RGD Ligand Internalization in Glioblastoma for MRI Application

    PubMed Central

    Moncelet, Damien; Bouchaud, Véronique; Mellet, Philippe; Ribot, Emeline; Miraux, Sylvain; Franconi, Jean-Michel; Voisin, Pierre

    2013-01-01

    Cellular density is a parameter measured for glioma grade and invasiveness diagnosis. The characterization of the cellular density can be performed, non invasively, by magnetic resonance imaging (MRI), since, this technique displays a good resolution. Nevertheless MRI sensitivity is critical. Development of smart contrast agents appears useful to increase MRI signal to noise ratio (SNR). Tumor invasiveness is correlated with high expression of integrins that can be targeted by RGD motif. In this study, MRI contrast agents or fluorescent probes linked to RGD-peptides were used, in a glioma model, to assess the relation between RGD uptake/signal improvement/cell density and consequently tumor invasiveness. Experiments were performed in vitro with U87-MG glioma cells. Flow cytometry and microscopy experiments with RGD and iRGD-alexa488 demonstrated that cell internalization was dependent on cell density. The internalization involved a clathrin-dependent endocytosis. Cytoskeleton and particularly the microtubules were concerned. Actin filaments played a minor role. The internalization was also dependent on the glycolysis and the oxidative phosphorylations. The cellular density modulated the importance of the endocytosis pathways and of the metabolism but not the cytoskeleton contribution. The internalization of the RGD-peptide associated to gadolinium chelate increased the SNR of U87 cells. Moreover, following the cell density augmentation, the SNR increased with a low amplitude but a trend was clearly determined. In conclusion, RGD-peptide internalization appeared, in vitro, as a marker of cellular density. In perspective, the combination of these peptides with contrast agents associated to more sensitive MRI techniques could improve the MRI signal allowing the characterization of cellular density for tumor diagnosis. PMID:24386117

  14. Impact of RGD amount in dextran-based hydrogels for cell delivery.

    PubMed

    Riahi, Nesrine; Liberelle, Benoît; Henry, Olivier; De Crescenzo, Gregory

    2017-04-01

    Dextran is one of the hydrophilic polymers that is used for hydrogel preparation. As any polysaccharide, it presents a high density of hydroxyl groups, which make possible several types of derivatization and crosslinking reactions. Furthermore, dextran is an excellent candidate for hydrogel fabrication with controlled cell/scaffold interactions as it is resistant to protein adsorption and cell adhesion. RGD peptide can be grafted to the dextran in order to promote selected cell adhesion and proliferation. Altogether, we have developed a novel strategy to graft the RGD peptide sequence to dextran-based hydrogel using divinyl sulfone as a linker. The resulting RGD functionalized dextran-based hydrogels were transparent, presented a smooth surface and were easy to handle. The impact of varying RGD peptide amounts, hydrogel porosity and topology upon human umbilical vein endothelial cell (HUVEC) adhesion, proliferation and infiltration was investigated. Our results demonstrated that 0.1% of RGD-modified dextran within the gel was sufficient to support HUVEC cells adhesion to the hydrogel surface. Sodium chloride was added (i) to the original hydrogel mix in order to form a macroporous structure presenting interconnected pores and (ii) to the hydrogel surface to create small orifices essential for cells migration inside the matrix.

  15. The effect of RGD density on osteoblast and endothelial cell behavior on RGD-grafted polyethylene terephthalate surfaces.

    PubMed

    Chollet, Celine; Chanseau, Christel; Remy, Murielle; Guignandon, Alain; Bareille, Reine; Labrugère, Christine; Bordenave, Laurence; Durrieu, Marie-C

    2009-02-01

    Hybrid materials combining polyethylene terephthalate and different types of cells (endothelial and osteoblastic cells) have been developed thanks to the covalent grafting of different densities of RGD containing peptides onto the polymer surface. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions, coupling agent grafting and the immobilization of the RGDC peptides. High resolution mu-imager was used to evaluate RGD densities (varying between 0.6 and 2.4 pmol/mm(2)) and has exhibited the stability of the surface grafted peptides when treated in harsh conditions. The efficiency of this route for biomimetic modification of a PET surface was demonstrated by measuring the adhesion of MC3T3 and HSVEC cells and by focal adhesion observation. Results obtained prove that a minimal RGDC density of 1 pmol/mm(2) is required to improve MC3T3 and HSVEC cells responses. Indeed, cells seeded onto a RGDC-modified PET with a density higher than 1 pmol/mm(2) were able to establish focal adhesion as visualized by fluorescence microscope compared to cells immobilized onto unmodified PET and RGDC-modified PET with densities lower than 1 pmol/mm(2). Moreover, the number of focal contacts was enhanced by the increase of RGDC peptide densities grafted onto the material surface. With this study we proved that the density of peptides immobilized on the surface is a very important parameter influencing osteoblast or endothelial cell adhesion and focal contact formation.

  16. RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vascular imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Belfield, Kevin D.; Yue, Xiling; Morales, Alma R.; Githaiga, Grace W.; Woodward, Adam W.; Tang, Simon; Sawada, Junko; Komatsu, Masanobu; Liu, Xuan

    2016-03-01

    Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 μm in tumor tissue.

  17. RGD-conjugated rod-like viral nanoparticles on 2D scaffold improved bone differentiation of mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Wang, Qian; Pongkwan, Sitasuwan; Lee, L.; Li, Kai; Nguyen, Huong

    2014-05-01

    Viral nanoparticles have uniform and well-defined nano-structures and can be produced in large quantities. Several plant viral nanoparticles have been tested in biomedical applications due to the lack of mammalian cell infectivity. We are particularly interested in using Tobacco mosaic virus (TMV), which has been demonstrated to enhance bone tissue regeneration, as a tuneable nanoscale building block for biomaterials development. Unmodified TMV particles have been shown to accelerate osteogenic differentiation of adult stem cells by synergistically upregulating BMP2 and IBSP expression with dexamethasone. However, the lack of affinity to mammalian cell surface resulted in low initial cell adhesion. In this study, to increase cell binding capacity of TMV based material the chemical functionalization of TMV with arginine-glycine-aspartic acid (RGD) peptide was explored. An azide-derivatized RGD peptide was “clicked” to tyrosine residues on TMV outer surface via an efficient copper(I) catalysed azide-alkyne cycloaddition reaction. The ligand spacing is calculated to be 2-4 nm, which could offer a polyvalent ligand clustering effect for enhanced cell receptor signalling, further promoting the proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells.

  18. RGD-conjugated Two-photon Absorbing Near-IR Emitting Fluorescent Probes for Tumor Vasculature Imaging

    PubMed Central

    Yue, Xiling; Morales, Alma R.; Githaiga, Grace W.; Woodward, Adam W.; Tang, Simon; Sawada, Junko; Komatsu, Masanobu; Liu, Xuan; Belfield, Kevin D.

    2015-01-01

    Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700–1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 μm in tumor tissue. PMID:26351137

  19. The Influence of RGD-Bearing Hydrogels on the Re-expression of Contractile Vascular Smooth Muscle Cell Phenotype

    PubMed Central

    Beamish, Jeffrey A.; Fu, Alexander; Choi, Ae-jin; Haq, Nada; Kottke-Marchant, Kandice; Marchant, Roger E.

    2009-01-01

    This study reports on the ability of poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with pendant integrin-binding GRGDSP peptides (RGD-gels) to support the re-differentiation of cultured vascular smooth muscle cells (SMCs) toward a contractile phenotype. Human coronary SMCs were seeded on RGD-gels, hydrogels with other extracellular matrix derived peptides, fibronectin (FN) and laminin (LN). Differentiation was induced on RGD-gels with low serum medium containing soluble heparin, and the differentiation status was monitored by mRNA expression, protein expression, and intracellular protein organization of the contractile smooth muscle markers, smooth muscle α-actin, calponin and SM-22α. RGD-gels supported a rapid induction (2.7- to 25-fold up-regulation) of SMC marker gene mRNA, with expression levels that were equivalent to FN and LN controls. Marker protein levels mirrored the changes in mRNA expression, with levels on RGD-gels indistinguishable from FN and LN controls. Furthermore, these markers co-localized in stress fibers within SMCs on RGD-gels suggesting the recapitulation of a contractile apparatus within the cells. These results indicate that SMCs cultured on RGD-bearing hydrogels can re-differentiate toward a contractile phenotype suggesting this material is an excellent candidate for further development as a bioactive scaffold that regulates SMC phenotype. PMID:19481795

  20. Histidine-lysine peptides as carriers of nucleic acids.

    PubMed

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

  1. Chiral separation of amino acids and peptides by capillary electrophoresis.

    PubMed

    Wan, H; Blomberg, L G

    2000-04-14

    Chiral separation of amino acids and peptides by capillary electrophoresis (CE) is reviewed regarding the separation principles of different approaches, advantages and limitations, chiral recognition mechanisms and applications. The direct approach details various chiral selectors with an emphasis on cyclodextrins and their derivatives, antibiotics and chiral surfactants as the chiral selectors. The indirect approach deals with various chiral reagents applied for diastereomer formation and types of separation media such as micelles and polymeric pseudo-stationary phases. Many derivatization reagents used for high sensitivity detection of amino acids and peptides are also discussed and their characteristics are summarized in tables. A large number of relevant examples is presented illustrating the current status of enantiomeric and diastereomeric separation of amino acids and peptides. Strategies to enhance the selectivity and optimize separation parameters by the application of experimental designs are described. The reversal of enantiomeric elution order and the effects of organic modifiers on the selectivity are illustrated in both direct and indirect methods. Some applications of chiral amino acid and peptide analysis, in particular, regarding the determination of trace enantiomeric impurities, are given. This review selects more than 200 articles published between 1988 and 1999.

  2. Peptide-directed binding of quantum dots to integrins in human fibroblast.

    PubMed

    Shi, Peng; Chen, Hongfeng; Cho, Michael R; Stroscio, Michael A

    2006-03-01

    There is currently a major international effort aimed at integrating semiconductor nanostructures with biological structures. This paper reports the use of peptide sequences with certain motifs like artinine-glycine-aspartic acid (RGD) and leucine-aspartic acid-valine (LDV) to functionalize zinc sulfide (ZnS)-capped cadmiun selenide (CdSe) quantum dots, so that the quantum dot-peptide complexes selectively bind to integrins on HT1080 human fibrosarcoma cells membrane. In this way, an interface between semiconductor nanocrystals and subcellular components was achieved, and the distribution pattern of RGD and LDV receptors on HT1080 cell membranes is revealed. These findings point the way to using a wide class of peptide-functionalized semiconductor quantum dots for the study of cellular processes involving integrins.

  3. cRGD-installed polymeric micelles loading platinum anticancer drugs enable cooperative treatment against lymph node metastasis.

    PubMed

    Makino, Jun; Cabral, Horacio; Miura, Yutaka; Matsumoto, Yu; Wang, Ming; Kinoh, Hiroaki; Mochida, Yuki; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2015-12-28

    Lymph node metastasis (LNM) is correlated with decreased survival, indicating high tumor malignancy and being a potential source for subsequent fatal metastases. Targeted therapies inhibiting the formation of LNM, while eliminating established metastatic foci, could provide synergistic effects by reducing the incidence and growth of metastasis. Based on the inhibitory activity of cRGD peptide against the development of metastasis, and the LNM targeting ability of systemically injected drug-loaded polymeric micelles, herein, we studied the capability of cRGD-installed polymeric micelles incorporating the platinum anticancer drug (1,2-diaminocylohexane)platinum(II) (DACHPt) for cooperatively inhibiting the formation and progression of LNM. As cRGD-installed DACHPt-loaded micelles (cRGD-DACHPt/m) presented similar size, drug loading and surface charge to non-conjugated micelles (MeO-DACHPt/m), the differences in the biological performance of the micelles were endorsed to the effect of the ligand. In a syngeneic melanoma model, both MeO-DACHPt/m and cRGD-DACHPt/m showed comparable antitumor activity against the primary tumors and the established metastatic foci in lymph nodes. However, cRGD-DACHPt/m significantly enhanced the efficacy against LNM draining from primary tumors through the effective inhibition of the spreading of cancer cells. This improved inhibition was associated with the ability of cRGD-DACHPt/m to reduce the migration of melanoma cells, which was higher than that of MeO-DACHPt/m, free cRGD and their combination. These results support our strategy of using cRGD-installed micelles for attaining cooperative therapies against LNM exploiting the inhibitory function of the peptide and the cytotoxic effect of the micelles.

  4. How Amino Acids and Peptides Shaped the RNA World

    PubMed Central

    van der Gulik, Peter T.S.; Speijer, Dave

    2015-01-01

    The “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a “pure RNA ribosome” evolution started out with. Though the oldest center of the ribosome seems “RNA only”, we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to basepairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the “RNA world” view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. In this article, we try to bring the role of the peptide component of early life back into focus. We argue that an RNA world completely independent of amino acids never existed. PMID:25607813

  5. Effects of RGD immobilization on light-induced cell sheet detachment from TiO2 nanodots films.

    PubMed

    Cheng, Kui; Wang, Tiantian; Yu, Mengliu; Wan, Hongping; Lin, Jun; Weng, Wenjian; Wang, Huiming

    2016-06-01

    Light-induced cell detachment is reported to be a safe and effective cell sheet harvest method. In the present study, the effects of arginine-glycine-aspartic acid (RGD) immobilization on cell growth, cell sheet construction and cell harvest through light illumination are investigated. RGD was first immobilized on TiO2 nanodots films through simple physical adsorption, and then mouse pre-osteoblastic MC3T3-E1 cells were seeded on the films. It was found that RGD immobilization promoted cell adhesion and proliferation. It was also observed that cells cultured on RGD immobilized films showed relatively high level of pan-cadherin. Cells harvested with ultraviolet illumination (365 nm) showed good viability on both RGD immobilized and unmodified TiO2 nanodot films. Single cell detachment assay showed that cells detached more quickly on RGD immobilized TiO2 nanodot films. That could be ascribed to the RGD release after UV365 illumination. The current study demonstrated that RGD immobilization could effectively improve both the cellular responses and light-induced cell harvest.

  6. cRGD-Modified Benzimidazole-based pH-Responsive Nanoparticles for Enhanced Tumor Targeted Doxorubicin Delivery.

    PubMed

    Liu, Jinjian; Liu, Qian; Yang, Cuihong; Sun, Yu; Zhang, Yumin; Huang, Pingsheng; Zhou, Junhui; Liu, Qiang; Chu, Liping; Huang, Fan; Deng, Liandong; Dong, Anjie; Liu, Jianfeng

    2016-05-04

    Finding a smart cancer drug delivery carrier with long blood circulation, enhanced cancer targeting, and quick drug release in tumors is critical for efficient cancer chemotherapy. Herein, we design a cRGD-polycarboxybetaine methacrylate-b-polybenzimidazole methacrylate (cRGD-PCB-b-PBBMZ) copolymer to self-assemble into smart drug-loaded nanoparticles (cRGD-PCM NPs) which can target αvβ3 integrin overexpressed cancer tissue by cRGD peptide unit and release drug quickly in cancer cells by protonation of benzimidazole groups. The outer PCB layer can resist protein adhesion, and there are only about 10% of proteins in mouse serum adhered to the surface of PCM NPs. With the pKa value of 5.08 of the benzimidazole units, DOX can be released from NPs in pH 5.0 PBS. cRGD-PCM NPs can bring more DOX into HepG2 cells than nontargeting PCM NPs, and there has high DOX release rate in HepG2 cells because of the protonation of benzimidazole groups in endosome and lysosome. MTT assay verifies that higher cellular uptake of DOX causes higher cytotoxicity. Furthermore, the results of ex vivo imaging studies confirm that cRGD-PCM/DOX NPs can successfully deliver DOX into tumor tissue from the injection site. Therefore, the multifunctional cRGD-PCM NPs show great potential as novel nanocarriers for targeting cancer chemotherapy.

  7. Characterization of peptide immobilization on an acetylene terminated surface via click chemistry

    NASA Astrophysics Data System (ADS)

    Shamsi, Fahimeh; Coster, Hans; Jolliffe, Katrina A.

    2011-10-01

    Peptide (A-A-A-A-G-G-G-E-R-G-D)1A: Alanine; D: Glutamic acid; E: Aspartic acid; G: Glycine; R: Arginine. conjugated surfaces were prepared on silicon surfaces through click chemistry. The amino acid sequence RGD is the cellular attachment site of a large number of extracellular matrices such as blood and cell surface proteins. Recent research has focused on developing RGD peptides which mimic cell adhesion proteins and integrins [1,2].The steps involved the formation of an alkyne-terminated monolayer on Si(111), followed by linking the peptide to 4-azidophenyl isothiocyanate via a specific and gentle reaction. This was followed by the attachment of the azido peptide to the surface-bound alkynes using the Cu (I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. The surface structures of the alkyne terminated monolayer and the attached peptide were characterized using high resolution impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS) and Fourier Transform Infrared (ATR-FTIR) Spectroscopy. EIS characterization revealed the alkyne layer and the hydrophobic and polar regions of the attached peptide. XPS analysis showed a high surface coverage of the peptide on the silicon substrates and this was confirmed by FTIR.Our results confirmed a specific covalent attachment of the peptide on the silicon surfaces. This approach offers a versatile, experimentally simple, method for the specific attachment of peptide ligands. This approach would have applications for cell attachment and biosensors.

  8. Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

    PubMed

    Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

    The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system.

  9. The antitumor activity of a doxorubicin loaded, iRGD-modified sterically-stabilized liposome on B16-F10 melanoma cells: in vitro and in vivo evaluation

    PubMed Central

    Yu, Ke-Fu; Zhang, Wei-Qiang; Luo, Li-Min; Song, Ping; Li, Dan; Du, Ruo; Ren, Wei; Huang, Dan; Lu, Wan-Liang; Zhang, Xuan; Zhang, Qiang

    2013-01-01

    Considering the fact that iRGD (tumor-homing peptide) demonstrates tumor-targeting and tumor-penetrating activity, and that B16-F10 (murine melanoma) cells overexpress both αv integrin receptor and neuropilin-1 (NRP-1), the purpose of this study was to prepare a novel doxorubicin (DOX)-loaded, iRGD-modified, sterically-stabilized liposome (SSL) (iRGD-SSL-DOX) in order to evaluate its antitumor activity on B16-F10 melanoma cells in vitro and in vivo. The iRGD-SSL-DOX was prepared using a thin-film hydration method. The characteristics of iRGD-SSL-DOX were evaluated. The in vitro leakage of DOX from iRGD-SSL-DOX was tested. The in vitro tumor-targeting and tumor-penetrating characteristics of iRGD-modified liposomes on B16-F10 cells were investigated. The in vivo tumor-targeting and tumor-penetrating activities of iRGD-modified liposomes were performed in B16-F10 tumor-bearing nude mice. The antitumor effect of iRGD-SSL-DOX was evaluated in B16-F10 tumor-bearing C57BL/6 mice in vivo. The average particle size of the iRGD-SSL-DOX was found to be 91 nm with a polydispersity index (PDI) of 0.16. The entrapment efficiency of iRGD-SSL-DOX was 98.36%. The leakage of DOX from iRGD-SSL-DOX at the 24-hour time point was only 7.5%. The results obtained from the in vitro flow cytometry and confocal microscopy, as well as in vivo biodistribution and confocal immunofluorescence microscopy experiments, indicate that the tumor-targeting and tumor-penetrating activity of the iRGD-modified SSL was higher than that of unmodified SSL. In vivo antitumor activity results showed that the antitumor effect of iRGD-SSL-DOX against melanoma tumors was higher than that of SSL-DOX in B16-F10 tumor-bearing mice. In conclusion, the iRGD-SSL-DOX is a tumor-targeting and tumor-penetrating peptide modified liposome which has significant antitumor activity against melanoma tumors. PMID:23885174

  10. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  11. Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas.

    PubMed

    Kawamura, Wataru; Miura, Yutaka; Kokuryo, Daisuke; Toh, Kazuko; Yamada, Naoki; Nomoto, Takahiro; Matsumoto, Yu; Sueyoshi, Daiki; Liu, Xueying; Aoki, Ichio; Kano, Mitsunobu R; Nishiyama, Nobuhiro; Saga, Tsuneo; Kishimura, Akihiro; Kataoka, Kazunori

    2015-06-01

    Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

  12. Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas

    NASA Astrophysics Data System (ADS)

    Kawamura, Wataru; Miura, Yutaka; Kokuryo, Daisuke; Toh, Kazuko; Yamada, Naoki; Nomoto, Takahiro; Matsumoto, Yu; Sueyoshi, Daiki; Liu, Xueying; Aoki, Ichio; Kano, Mitsunobu R.; Nishiyama, Nobuhiro; Saga, Tsuneo; Kishimura, Akihiro; Kataoka, Kazunori

    2015-06-01

    Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

  13. Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas

    PubMed Central

    Kawamura, Wataru; Miura, Yutaka; Kokuryo, Daisuke; Toh, Kazuko; Yamada, Naoki; Nomoto, Takahiro; Matsumoto, Yu; Sueyoshi, Daiki; Liu, Xueying; Aoki, Ichio; Kano, Mitsunobu R; Nishiyama, Nobuhiro; Saga, Tsuneo; Kishimura, Akihiro; Kataoka, Kazunori

    2015-01-01

    Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins. PMID:27877805

  14. Quaternary Zn-Ag-In-Se quantum dots for biomedical optical imaging of RGD-modified micelles.

    PubMed

    Deng, Dawei; Qu, Lingzhi; Zhang, Jian; Ma, Yuxiang; Gu, Yueqing

    2013-11-13

    Exploring the synthesis of new biocompatible quantum dots (QDs) helps in overcoming the intrinsic toxicity of the existing QDs composed of highly toxic heavy metals (e.g., Cd, Hg, Pb, etc.) and is particularly interesting for the future practical application of QDs in biomedical imaging. Hence, in this report, a new one-pot approach to oil-soluble (highly toxic heavy metal-free) highly luminescent quaternary Zn-Ag-In-Se (ZAISe) QDs was designed. Their photoluminescence (PL) emission could be systematically tuned from 660 to 800 nm by controlling the Ag/Zn feed ratio, and their highest PL quantum yield is close to 50% after detailed optimization. Next, by using biodegradable RGD peptide (arginine-glycine-aspartic acid)-modified N-succinyl-N'-octyl-chitosan (RGD-SOC) micelles as a water transfer agent, the versatility of these quaternary ZAISe QDs for multiscale bioimaging of micelles (namely, in vitro and in vivo evaluating the tumor targeting of drug carriers) was further explored, as a promising alternative for Cd- and Pb-based QDs.

  15. Encapsulation of eptifibatide in RGD-modified nanoliposomes improves platelet aggregation inhibitory activity.

    PubMed

    Bardania, Hassan; Shojaosadati, Seyed Abbas; Kobarfard, Farzad; Dorkoosh, Farid; Zadeh, Marjan Esfahani; Naraki, Mahmoud; Faizi, Mehrdad

    2017-02-01

    Eptifibatide is an antiplatelet drug used for the treatment of thrombosis. However, as a result of its accumulation in non-targeted tissues and short half-life, it has a limited efficacy. In this study, RGD-modified nano-liposomes (RGD-MNL) were prepared as carriers for the targeted delivery of eptifibatide to activated platelets. The nano-liposomes were about 90 ± 10 nm in size, with an encapsulation efficiency of 37 ± 5 % and a good stability during 21 days, with a negligible change in the size of nanoliosomes. The in vitro cytotoxicity of nanoliposomes was examined using MTT assay. The results obtained from the ex vivo study showed that the antiplatelet activity of eptifibatide encapsulated nanoliposomes was higher in comparison with the free drug (81.63 vs. 46.17 % for RGD-MNL) and (66.67 vs. 46.17 % for UNL), and this increase was more significant for nanoliposomes targeted with RGD peptide (81.63 %; p < 0.05). The results indicated that RGD-MNL encapsulated eptifibatide had no significant cytotoxic effect on cells. In conclusion, the present nanoliposome formulation can be regarded as a new delivery system for protection and enhancement of the antiplatelet activity of eptifibatide.

  16. Recombinant decorsin: dynamics of the RGD recognition site.

    PubMed Central

    Krezel, A. M.; Ulmer, J. S.; Wagner, G.; Lazarus, R. A.

    2000-01-01

    Decorsin is an antagonist of integrin alphaIIbbeta3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size-exclusion filtration of the periplasmic contents followed by reversed-phase high-performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate b-methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities. PMID:10975565

  17. Kojic Acid Peptide: A New Compound with Anti-Tyrosinase Potential

    PubMed Central

    Singh, Birendra Kumar; Park, Seok Hoon; Lee, Hyang-Bok; Goo, Young-Aae; Kim, Hyoung Shik; Cho, Seung Hee; Lee, Jeong Hun; Ahn, Ghe Whan; Kim, Jin Pyo; Kang, Su Myoung

    2016-01-01

    Background Kojic acid was used for decades in the cosmetic industry as an antimelanogenic agent. However, there are two major drawbacks of Kojic acid, one is cytotoxicity and second are instability on storage. These limitations led the scientist to synthesize the active Kojic acid peptides. Objective In the present study, we synthesize and investigate the effect of five Kojic acid peptides to overcome the limitation of Kojic acid. Methods The peptide was analyzed and purified by high-performance liquid chromatography and matrix-assisted laser desorption ionization time of flight mass spectroscopy. Further, the tyrosinase activities of the Kojic acid and Kojic acid peptides were compared. The toxicity was measured and the melanin content is recorded in B16F10 mouse melanoma cells. Results Maximum tyrosinase activity was measured by Kojic acid peptides. Therefore, Kojic acid peptides were subjected to melanin assay and cytotoxicity assay and finally the stability of the Kojic acid peptide was measured. Conclusion It was observed that this newly synthesized Kojic acid peptide is stable and potent to inhibit the tyrosinase activity and melanin content of B16F10 mouse melanoma cells without exhibiting cell toxicity. Together, these preliminary results suggest that a further exploration is being needed to establish Kojic acid peptide as antimelanogenic agent. PMID:27746633

  18. The coxsackievirus A9 RGD motif is not essential for virus viability.

    PubMed Central

    Hughes, P J; Horsnell, C; Hyypiä, T; Stanway, G

    1995-01-01

    An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization. PMID:7494317

  19. The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides

    PubMed Central

    Sophocleous, Andreas M.; Desai, Kashappa-Goud H.; Mazzara, J. Maxwell; Tong, Ling; Cheng, Ji-Xin; Olsen, Karl F.; Schwendeman, Steven P.

    2013-01-01

    An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2-4 mM octreotide or leuprolide acetate salts in 0.1 M HEPES buffer, pH 7.4, with polymer particles or films at 4-37 °C for 24 h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy and stimulated Raman scattering (SRS) and laser scanning confocal imaging techniques were used to examine peptide penetration in the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST + 0.02% sodium azide, 37 °C) and in vivo (male Sprague-Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but can also internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~17 wt% leuprolide loading in the polymer (i.e., ~70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for > 2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague-Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides. PMID:24021356

  20. Peri-implant bone formation and implant integration strength of peptide-modified p(AAM-co-EG/AAC) interpenetrating polymer network-coated titanium implants.

    PubMed

    Barber, Thomas A; Ho, James E; De Ranieri, Aladino; Virdi, Amarjit S; Sumner, Dale R; Healy, Kevin E

    2007-02-01

    Interpenetrating polymer networks (IPNs) of poly (acrylamide-co-ethylene glycol/acrylic acid) functionalized with an -Arg-Gly-Asp- (RGD) containing 15 amino acid peptides, derived from rat bone sialoprotein (bsp-RGD(15), were grafted to titanium implants in an effort to modulate bone formation in the peri-implant region in the rat femoral ablation model. Bone-implant contact (BIC) and bone formation within the medullary canal were determined using microcomputed tomography at 2 and 4 weeks postimplantation. BIC for bsp-RGD(15)-IPN implants was enhanced relative to hydroxyapatite tricalcium phosphate (HA-TCP) coated implants, but was similar to all other groups. Aggregate bone formation neither indicated a dose-dependent effect of bsp-RGD(15) nor a meaningful trend. Mechanical testing of implant fixation revealed that only the HA-TCP coated implants supported significant (>1 MPa) interfacial shear strength, despite exhibiting lower overall BIC, an indication that bone ingrowth into the rougher coating was the primary mode of implant fixation. While no evidence was found to support the hypothesis that bsp-RGD(15)-modified IPN coated implants significantly impacted bone-implant bonding, these results point to the lack of correlation between in vitro studies employing primary osteoblasts and in vivo wound healing in the peri-implant region.

  1. Molecular self-assembly using peptide nucleic acids.

    PubMed

    Berger, Or; Gazit, Ehud

    2017-01-01

    Peptide nucleic acids (PNAs) are extensively studied for the control of genetic expression since their design in the 1990s. However, the application of PNAs in nanotechnology is much more recent. PNAs share the specific base-pair recognition characteristic of DNA together with material-like properties of polyamides, both proteins and synthetic polymers, such as Kevlar and Nylon. The first application of PNA was in the form of PNA-amphiphiles, resulting in the formation of either lipid integrated structures, hydrogels or fibrillary assemblies. Heteroduplex DNA-PNA assemblies allow the formation of hybrid structures with higher stability as compared with pure DNA. A systematic screen for minimal PNA building blocks resulted in the identification of guanine-containing di-PNA assemblies and protected guanine-PNA monomer spheres showing unique optical properties. Finally, the co-assembly of PNA with thymine-like three-faced cyanuric acid allowed the assembly of poly-adenine PNA into fibers. In summary, we believe that PNAs represent a new and important family of building blocks which converges the advantages of both DNA- and peptide-nanotechnologies.

  2. Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence

    PubMed Central

    Moreira, Susana M; Andrade, Fábia K; Domingues, Lucíla; Gama, Miguel

    2008-01-01

    Background Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an α-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. Results The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. Conclusion The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications. PMID:18854017

  3. Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma.

    PubMed

    Yandle, T; Crozier, I; Nicholls, G; Espiner, E; Carne, A; Brennan, S

    1987-07-31

    Two atrial natriuretic peptides were purified from pooled human coronary sinus plasma by Sep-Pak extraction, immunoaffinity chromatography and reverse phase HPLC. The amino acid sequences of the two peptides were homologous with 99-126 human atrial natriuretic peptide (hANP) and 106-126 hANP, the latter being most probably linked to 99-105 ANP by the disulphide bond. The molar ratio of the peptides in plasma, as assessed by radioimmunoassay was 10:3.

  4. An iRGD Based Strategy to Study Electrochemically the Species Inside a Cell

    PubMed Central

    Ning, Limin; Li, Xiaoxi; Ding, Xiaorong; Yin, Yongmei; Li, Genxi

    2012-01-01

    This paper reports a method for electrical communication between the inner part of cells and an electrode with the help of iRGD peptide. Due to the enhancement of the cell penetration caused by iRGD peptide, DNA molecules, previously modified on a gold electrode surface, can be easily transfected into the cells. At the same time, doxorubicin, an anticancer drug, can also be transfected into cells with high penetration. Consequently, doxorubicin binds to DNA chains through electrostatic interaction, and the redox reaction is transferred out of the cell across the cell membrane. As a result, this work may provide a novel way to get information from inside of cells. PMID:22949871

  5. Bio-compatibility of ion beam-modified and RGD-grafted polyethylene

    NASA Astrophysics Data System (ADS)

    Ročková-Hlaváčková, K.; Švorčík, V.; Bačáková, L.; Dvořánková, B.; Heitz, J.; Hnatowicz, V.

    2004-09-01

    Polyethylene (PE) was implanted with 15 keV Ar + and Kr + ions to the fluences from 3 × 10 12-1 × 10 15 cm -2 and subsequently grafted with amino acid sequence Arg-Gly-Asp (RGD), i.e. a minimum adhesion motif recognized by integrin receptors on cells. The structural changes of PE were studied using goniometric technique, UV-VIS, LIF spectroscopy and by measuring specimen electrical resistance. The adhesion and proliferation of mouse embryonic fibroblasts 3T3 on the modified PE were studied under in vitro conditions. Addition of RGD sequence onto double bonds created by the ion irradiation on polymeric chain was observed. The adhesion and proliferation of the 3T3 cells is increased by ion implantation and additionally also by RGD grafting.

  6. Di-heterometalation of thiol-functionalized peptide nucleic acids

    PubMed Central

    Joshi, Tanmaya; Patra, Malay; Spiccia, Leone; Gasser, Gilles

    2013-01-01

    As a proof-of-principle, two hetero-bimetallic PNA oligomers containing a ruthenium(II) polypyridyl and a cyclopentadienyl manganese tricarbonyl complex have been prepared by serial combination of solid-phase peptide coupling and in-solution thiol chemistry. Solid-phase N-terminus attachment of Ru(II)-polypyridyl carboxylic acid derivative, C1, onto the thiol-functionalized PNA backbone (H-a-a-g-t-c-t-g-c-linker-cys-NH2) has been performed by standard peptide coupling method. As two parallel approaches, the strong affinity of thiols for maleimide and haloacetyl group has been exploited for subsequent post-SPPS addition of cymantrene-based organometallic cores, C2 and C3. Michael-like addition and thioether ligation of thiol functionalized PNA1 (H-gly-a-a-g-t-c-t-g-c-linker-cys-NH2) and PNA2 (C1-a-a-g-t-c-t-g-c-linker-cys-NH2) to cymantrene maleimide and chloroacetyl derivatives, C2 and C3, respectively, has been performed. The synthesized ruthenium(II)-cymantrenyl PNA oligomers have been characterized by mass spectrometry (ESI-MS) and IR spectroscopy. The distinct Mn-CO vibrational IR stretches, between 1,924–2,074 cm−1, have been used as markers to confirm the presence of cymantrenyl units in the PNA sequences and the purity of the HPLC-purified PNA thioethers assessed using LC-MS. PMID:23422249

  7. Synthesis of hybrid hydrazino peptides: protected vs unprotected chiral α-hydrazino acids.

    PubMed

    Suć, Josipa; Jerić, Ivanka

    2015-01-01

    Peptidomimetics based on hydrazino derivatives of α-amino acids represent an important class of peptidic foldamers with promising biological activities, like protease inhibition and antimicrobial activity. However, the lack of straightforward method for the synthesis of optically pure hydrazino acids and efficient incorporation of hydrazino building blocks into peptide sequence hamper wider exploitation of hydrazino peptidomimetics. Here we described the utility of N (α)-benzyl protected and unprotected hydrazino derivatives of natural α-amino acids in synthesis of peptidomimetics. While incorporation of N (α)-benzyl-hydrazino acids into peptide chain and deprotection of benzyl moiety proceeded with difficulties, unprotected hydrazino acids allowed fast and simple construction of hybrid peptidomimetics.

  8. Pyrrolidinyl peptide nucleic acid homologues: effect of ring size on hybridization properties.

    PubMed

    Mansawat, Woraluk; Vilaivan, Chotima; Balázs, Árpád; Aitken, David J; Vilaivan, Tirayut

    2012-03-16

    The effect of ring size of four- to six-membered cyclic β-amino acid on the hybridization properties of pyrrolidinyl peptide nucleic acid with an alternating α/β peptide backbone is reported. The cyclobutane derivatives (acbcPNA) show the highest T(m) and excellent specificity with cDNA and RNA.

  9. New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations.

    PubMed

    Melnykov, Artem V

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.

  10. Desalted duck egg white peptides promote calcium uptake by counteracting the adverse effects of phytic acid.

    PubMed

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

    2017-03-15

    The structure of the desalted duck egg white peptides-calcium chelate was characterized by fluorescence spectroscopy, fourier transform infrared spectroscopy, and dynamic light scattering. Characterization results showed structural folding and aggregation of amino acids or oligopeptides during the chelation process. Desalted duck egg white peptides enhanced the calcium uptake in the presence of oxalate, phosphate and zinc ions in Caco-2 monolayers. Animal model indicated that desalted duck egg white peptides effectively enhanced the mineral absorption and counteracted the deleterious effects of phytic acid. These findings suggested that desalted duck egg white peptides might promote calcium uptake in three pathways: 1) desalted duck egg white peptides bind with calcium to form soluble chelate and avoid precipitate; 2) the chelate is absorbed as small peptides by enterocyte; and 3) desalted duck egg white peptides regulate the proliferation and differentiation of enterocytes through the interaction with transient receptor potential vanilloid 6 calcium channel.

  11. Comparative studies of adhesion peptides based on l- or d-amino acids.

    PubMed

    Nikitin, Sergey; Palmer, Daniel; Meldal, Morten; Diness, Frederik

    2016-10-01

    Detailed studies comparing solid-supported l- or d-amino acid adhesion peptides based on the sequence KLHRIRA were performed. Stability towards proteases and levels of cellular adhesion to the otherwise inert surface of PEGA resin were compared by using fluorescently labelled peptides. A clear difference in the peptide stability towards cleavage by subtilisin, trypsin, or papain was observed. However, all of the on-bead peptides provided an optimal surface for cell adhesion and proliferation. In long-term experiments, these properties were still found to be similar on the resins modified either with l- or with d-amino acids and unaffected by the nature of their fluorescence labelling at either terminus. These results support that the more accessible l-amino acids can be utilized for cell adhesion experiments and confirm the nonspecific interaction mechanism of cell binding to these peptides on the bead surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  12. Poly(amino acid)-based fibrous scaffolds modified with surface-pendant peptides for cartilage tissue engineering.

    PubMed

    Svobodová, Jana; Proks, Vladimír; Karabiyik, Özge; Çalıkoğlu Koyuncu, Ayse Ceren; Torun Köse, Gamze; Rypáček, František; Studenovská, Hana

    2017-03-01

    In this study, fibrous scaffolds based on poly(γ-benzyl-l-glutamate) (PBLG) were investigated in terms of the chondrogenic differentiation potential of human tooth germ stem cells (HTGSCs). Through the solution-assisted bonding of the fibres, fully connected scaffolds with pore sizes in the range 20-400 µm were prepared. Biomimetic modification of the PBLG scaffolds was achieved by a two-step reaction procedure: first, aminolysis of the PBLG fibres' surface layers was performed, which resulted in an increase in the hydrophilicity of the fibrous scaffolds after the introduction of N(5) -hydroxyethyl-l-glutamine units; and second, modification with the short peptide sequence azidopentanoyl-GGGRGDSGGGY-NH2 , using the 'click' reaction on the previously modified scaffold with 2-propynyl side-chains, was performed. Radio-assay of the (125) I-labelled peptide was used to evaluate the RGD density in the fibrous scaffolds (which varied in the range 10(-3) -10 pm/cm(2) ). All the PBLG scaffolds, especially with density 90 ± 20 fm/cm(2) and 200 ± 100 fm/cm(2) RGD, were found to be potentially suitable for growth and chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

    PubMed Central

    Polverino de Laureto, P.; Scaramella, E.; De Filippis, V.; Marin, O.; Doni, M. G.; Fontana, A.

    1998-01-01

    Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal

  14. Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

    PubMed

    Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

    2017-01-01

    Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by (1)H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

  15. VE-cadherin RGD motifs promote metastasis and constitute a potential therapeutic target in melanoma and breast cancers

    PubMed Central

    Bartolomé, Rubén A.; Torres, Sofía; de Val, Soledad Isern; Escudero-Paniagua, Beatriz; Calviño, Eva; Teixidó, Joaquín; Casal, J. Ignacio

    2017-01-01

    We have investigated the role of vascular-endothelial (VE)-cadherin in melanoma and breast cancer metastasis. We found that VE-cadherin is expressed in highly aggressive melanoma and breast cancer cell lines. Remarkably, inactivation of VE-cadherin triggered a significant loss of malignant traits (proliferation, adhesion, invasion and transendothelial migration) in melanoma and breast cancer cells. These effects, except transendothelial migration, were induced by the VE-cadherin RGD motifs. Co-immunoprecipitation experiments demonstrated an interaction between VE-cadherin and α2β1 integrin, with the RGD motifs found to directly affect β1 integrin activation. VE-cadherin-mediated integrin signaling occurred through specific activation of SRC, ERK and JNK, including AKT in melanoma. Knocking down VE-cadherin suppressed lung colonization capacity of melanoma or breast cancer cells inoculated in mice, while pre-incubation with VE-cadherin RGD peptides promoted lung metastasis for both cancer types. Finally, an in silico study revealed the association of high VE-cadherin expression with poor survival in a subset of melanoma patients and breast cancer patients showing low CD34 expression. These findings support a general role for VE-cadherin and other RGD cadherins as critical regulators of lung and liver metastasis in multiple solid tumours. These results pave the way for cadherin-specific RGD targeted therapies to control disseminated metastasis in multiple cancers. PMID:27966446

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

    NASA Astrophysics Data System (ADS)

    Suwannachot, Yuttana; Rode, Bernd M.

    1999-10-01

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

  17. Isolation and nature of intracellular alpha-aminoadipic acid-containing peptides from Paecilomyces persicinus P-10.

    PubMed Central

    Eriquez, L A; Pisano, M A

    1979-01-01

    Small intracellular peptides containing alpha-aminoadipic acid, cysteine, and a valine moiety were obtained from mycelia of Paecilomyces persicinus P-10 by ethanol or trichloroacetic acid extraction. After performic acid oxidation and ion-exchange chromatography, analysis of the peptide fractions by two-dimensional thin-layer electrophoresis and chromatography revealed the presence of three related peptides, as sulfonic acid derivatives, each containing alpha-aminoadipic acid. Each peptide was isolated in chromatographically pure form by semipreparative thin-layer electrophoresis and chromatography. The purified peptides were subjected to differential hydrolysis, dansylation, and combined dansylation-phenylisothiocyanate sequence analysis. Based on these studies, the structures of the isolated peptides were determined to be (i) glycl-delta-(alpha-aminoadipyl)-cysteinyl-beta-hydroxyvaline, (ii) glycyl-delta-(alpha-aminoadipyl)-cysteinylvaline, and (iii) delta-(alpha-aminoadipyl)-cysteinylvaline. The peptides isolated from Paecilomyces are similar to the alpha-aminoadipic acid-cysteine-valine moiety complex peptides isolated from Cephalosporium. PMID:574371

  18. Adhesive and Migratory Effects of Phosphophoryn Are Modulated by Flanking Peptides of the Integrin Binding Motif

    PubMed Central

    Suzuki, Shigeki; Kobuke, Seiji; Haruyama, Naoto; Hoshino, Hiroaki; Kulkarni, Ashok B.; Nishimura, Fusanori

    2014-01-01

    Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  19. Phospholipid conjugate for intracellular delivery of peptide nucleic acids

    PubMed Central

    Shen, Gang; Fang, Huafeng; Song, Yinyin; Bielska, Agata A.; Wang, Zhenghui; Taylor, John-Stephen A.

    2009-01-01

    Peptide nucleic acids (PNAs) have a number of attractive features that have made them an ideal choice for antisense and antigene-based tools, probes and drugs, but their poor membrane permeability has limited their application as therapeutic or diagnostic agents. Herein we report a general method for the synthesis of phospholipid-PNAs (LP-PNAs), and compare the effect of non-cleavable lipids and bioreductively cleavable lipids (L and LSS) and phospholipid (LP) on the splice-correcting bioactivity of a PNA bearing the cell penetrating Arg9 group (PNA-R9). While the three constructs show similar and increasing bioactivity at 1–3 μM, the activity of LP-PNA-R9 continues to increase from 4–6 μM while the activity of L-PNA-R9 remains constant and LSS-PNA-R9 decreases rapidly in parallel with their relative cytotoxicity. The activity of both LP-PNA-R9 and L-PNA-R9 were found to dramatically increase with chloroquine, as expected for an endocytotic entry mechanism. Both constructs were also found to have CMC values of 1.0 and 4.5 μM in 150 mM NaCl, pH 7 water, suggesting that micelle formation may play a hitherto unrecognized role in modulating toxicity and/or facilitating endocytosis. PMID:19678628

  20. Surface investigation on biomimetic materials to control cell adhesion: the case of RGD conjugation on PCL.

    PubMed

    Causa, Filippo; Battista, Edmondo; Della Moglie, Raffaella; Guarnieri, Daniela; Iannone, Maria; Netti, Paolo A

    2010-06-15

    The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(epsilon-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates.

  1. Supramolecular control of self-assembling terthiophene-peptide conjugates through the amino acid side chain

    SciTech Connect

    Lehrman, Jessica A.; Cui, Honggang; Tsai, Wei-Wen; Moyer, Tyson J.; Stupp, Samuel I.

    2013-07-30

    The self-assembly of oligothiophene–peptide conjugates can be directed through the systematic variation of the peptide sequence into different nanostructures, including flat spicules, nanotubes, spiral sheets, and giant, flat sheets. Furthermore, the assembly of these molecules is not controlled by steric interactions between the amino acid side chains.

  2. Noninvasive monitoring of early antiangiogenic therapy response in human nasopharyngeal carcinoma xenograft model using MRI with RGD-conjugated ultrasmall superparamagnetic iron oxide nanoparticles

    PubMed Central

    Cui, Yanfen; Zhang, Caiyuan; Luo, Ran; Liu, Huanhuan; Zhang, Zhongyang; Xu, Tianyong; Zhang, Yong; Wang, Dengbin

    2016-01-01

    Purpose Arginine-glycine-aspartic acid (RGD)-based nanoprobes allow specific imaging of integrin αvβ3, a protein overexpressed during angiogenesis. Therefore, this study applied a novel RGD-coupled, polyacrylic acid (PAA)-coated ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-PAA-USPIO) in order to detect tumor angiogenesis and assess the early response to antiangiogenic treatment in human nasopharyngeal carcinoma (NPC) xenograft model by magnetic resonance imaging (MRI). Materials and methods The binding specificity of RGD-PAA-USPIO with human umbilical vein endothelial cells (HUVECs) was confirmed by Prussian blue staining and transmission electron microscopy in vitro. The tumor targeting of RGD-PAA-USPIO was evaluated in the NPC xenograft model. Later, mice bearing NPC underwent MRI at baseline and after 4 and 14 days of consecutive treatment with Endostar or phosphate-buffered saline (n=10 per group). Results The specific uptake of the RGD-PAA-USPIO nanoparticles was mainly dependent on the interaction between RGD and integrin αvβ3 of HUVECs. The tumor targeting of RGD-PAA-USPIO was observed in the NPC xenograft model. Moreover, the T2 relaxation time of mice in the Endostar-treated group decreased significantly compared with those in the control group both on days 4 and 14, consistent with the immunofluorescence results of CD31 and CD61 (P<0.05). Conclusion This study demonstrated that the magnetic resonance molecular nanoprobes, RGD-PAA-USPIOs, allow noninvasive in vivo imaging of tumor angiogenesis and assessment of the early response to antiangiogenic treatment in NPC xenograft model, favoring its potential clinical translation. PMID:27895477

  3. Cyclic Sulfamidate Enabled Syntheses of Amino Acids, Peptides, Carbohydrates, and Natural Products

    EPA Science Inventory

    This article reviews the emergence of cyclic sulfamidates as versatile intermediatesfor the synthesis of unnatural amino acids, chalcogen peptides, modified sugars, drugs and drug candidates, and important natural products.

  4. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    PubMed

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

  5. The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function

    PubMed Central

    Larsen, Coby C.; Kligman, Faina; Kottke-Marchant, Kandice; Marchant, Roger E.

    2007-01-01

    We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on ePTFE vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120° to 6°, indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function. PMID:16762410

  6. The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

    PubMed

    Larsen, Coby C; Kligman, Faina; Kottke-Marchant, Kandice; Marchant, Roger E

    2006-10-01

    We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.

  7. Systematic studies of the mass spectrometric properties of alkaline earth metal cationized amino acids and peptides

    NASA Astrophysics Data System (ADS)

    Küjckelmann, Ulrich; Müller, Dietrich; Weber, Carsten

    1997-07-01

    The results of a systematic study of the gas phase interactions of α-amino acids and peptides (4-15 amino acids) with alkaline earth metals, observed with mass spectrometric techniques, are presented. Furthermore, a model for the cationization with calcium at the C-terminal amino acid arginine in rotaviral polypeptides is presented.

  8. Increased antitumor activity of tumor-specific peptide modified thymopentin.

    PubMed

    Lao, Xingzhen; Li, Bin; Liu, Meng; Chen, Jiao; Gao, Xiangdong; Zheng, Heng

    2014-12-01

    Thymopoietin pentapeptide (thymopentin, TP5), an immunomodulatory peptide, has been successfully used as an immune system enhancer for treating immune deficiency, cancer, and infectious diseases. However, poor penetration into tumors remains a key limitation to the efficacy and application of TP5. iRGD (CRGDK/RGPD/EC) has been introduced to certain anticancer agents, and increased specific tumor penetrability of drugs and cell internalization have been observed. In the present study, we fused this iRGD fragment with the C-terminal of TP5 to yield a new product, TP5-iRGD. Cell attachment assay showed that TP5-iRGD exhibits more extensive attachment to the melanoma cell line B16F10 than wild-type TP5. Tumor cell viability assay showed that iRGD conjugation with the TP5 C-terminus increases the basal antiproliferative activity of the pentapeptide against the melanoma cell line B16F10, the human lung cancer cell line H460, and the human breast cancer cell line MCF-7. Subsequent injections of TP5-iRGD inhibited in vivo melanoma progression more efficiently than the native TP5. Murine spleen lymphocyte proliferation assay also showed that TP5-iRGD and the parent pentapeptide feature nearly identical spleen lymphocyte proliferation activities. We built an integrin αvβ3 and TP5-iRGD computational binding model to investigate the mechanism by which TP5-iRGD promotes increased activity further. Conjugation with iRGD promotes binding to integrin αvβ3, thereby increasing the tumor-homing efficiency of the resultant peptide. These experimental and computational observations of increased TP5-iRGD activity help broaden the usage of TP5 and reflect the great application potential of the peptide as an anticancer agent.

  9. Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

    PubMed

    Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

    2016-04-01

    Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time.

  10. Antimicrobial Peptides Containing Unnatural Amino Acid Exhibit Potent Bactericidal Activity against ESKAPE Pathogens

    DTIC Science & Technology

    2013-01-01

    Antimicrobial peptides containing unnatural amino acid exhibit potent bactericidal activity against ESKAPE pathogens R. P. Hicks a, J. J. Abercrombie...tic classes, membrane-disruptors and non -membrane-disrup- tors.30,31 Five different mechanisms have been proposed at one time or another to explain...DATES COVERED - 4. TITLE AND SUBTITLE Antimicrobial Peptides Containing Unnatural Amino Acid Exhibit Potent Bactericidal Activity Against

  11. Simultaneous separation of acid and basic bioactive peptides by electrodialysis with ultrafiltration membrane.

    PubMed

    Poulin, Jean-François; Amiot, Jean; Bazinet, Laurent

    2006-05-29

    beta-Lactoglobulin (beta-lg), one of the major whey components, can release by enzymatic hydrolysis different bioactive peptidic sequences according to the enzyme used. However, these protein hydrolysates have to be fractionated to obtain peptides in a more purified form. The aim of the present work was to evaluate the feasibility of separating peptides from a beta-lg hydrolysate using an ultrafiltration (UF) membrane stacked in an electrodialysis (ED) cell and to study the effect of pH on the migration of basic/cationic and acid/anionic peptides in the ED configuration. Electrodialysis with ultrafiltration membrane (EDUF) appeared to be a selective method of separation since amongst a total of 40 peptides in the raw hydrolysate, only 13 were recovered in the separated adjacent solutions (KCl 1 and KCl 2). Amongst these 13 migrating peptides, 3 acid/anionic peptides migrated only in one compartment (KCl 1), while 3 basic/cationic peptides migrated only in the second compartment (KCl 2) and that whatever the pH conditions of the hydrolysate solution. Furthermore, the highest migration was obtained for the ACE-inhibitory peptide beta-lg 142-148, with a value of 10.75%. The integrity of the UF membrane was kept and EDUF would minimize the fouling of UF membrane.

  12. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    PubMed

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  13. RGD-conjugated silica-coated gold nanorods on the surface of carbon nanotubes for targeted photoacoustic imaging of gastric cancer

    NASA Astrophysics Data System (ADS)

    Wang, Can; Bao, Chenchen; Liang, Shujing; Fu, Hualin; Wang, Kan; Deng, Min; Liao, Qiande; Cui, Daxiang

    2014-05-01

    Herein, we reported for the first time that RGD-conjugated silica-coated gold nanorods on the surface of multiwalled carbon nanotubes were successfully used for targeted photoacoustic imaging of in vivo gastric cancer cells. A simple strategy was used to attach covalently silica-coated gold nanorods (sGNRs) onto the surface of multiwalled carbon nanotubes (MWNTs) to fabricate a hybrid nanostructure. The cross-linked reaction occurred through the combination of carboxyl groups on the MWNTs and the amino group on the surface of sGNRs modified with a silane coupling agent. RGD peptides were conjugated with the sGNR/MWNT nanostructure; resultant RGD-conjugated sGNR/MWNT probes were investigated for their influences on viability of MGC803 and GES-1 cells. The nude mice models loaded with gastric cancer cells were prepared, the RGD-conjugated sGNR/MWNT probes were injected into gastric cancer-bearing nude mice models via the tail vein, and the nude mice were observed by an optoacoustic imaging system. Results showed that RGD-conjugated sGNR/MWNT probes showed good water solubility and low cellular toxicity, could target in vivo gastric cancer cells, and obtained strong photoacoustic imaging in the nude model. RGD-conjugated sGNR/MWNT probes will own great potential in applications such as targeted photoacoustic imaging and photothermal therapy in the near future.

  14. RGD-conjugated silica-coated gold nanorods on the surface of carbon nanotubes for targeted photoacoustic imaging of gastric cancer.

    PubMed

    Wang, Can; Bao, Chenchen; Liang, Shujing; Fu, Hualin; Wang, Kan; Deng, Min; Liao, Qiande; Cui, Daxiang

    2014-01-01

    Herein, we reported for the first time that RGD-conjugated silica-coated gold nanorods on the surface of multiwalled carbon nanotubes were successfully used for targeted photoacoustic imaging of in vivo gastric cancer cells. A simple strategy was used to attach covalently silica-coated gold nanorods (sGNRs) onto the surface of multiwalled carbon nanotubes (MWNTs) to fabricate a hybrid nanostructure. The cross-linked reaction occurred through the combination of carboxyl groups on the MWNTs and the amino group on the surface of sGNRs modified with a silane coupling agent. RGD peptides were conjugated with the sGNR/MWNT nanostructure; resultant RGD-conjugated sGNR/MWNT probes were investigated for their influences on viability of MGC803 and GES-1 cells. The nude mice models loaded with gastric cancer cells were prepared, the RGD-conjugated sGNR/MWNT probes were injected into gastric cancer-bearing nude mice models via the tail vein, and the nude mice were observed by an optoacoustic imaging system. Results showed that RGD-conjugated sGNR/MWNT probes showed good water solubility and low cellular toxicity, could target in vivo gastric cancer cells, and obtained strong photoacoustic imaging in the nude model. RGD-conjugated sGNR/MWNT probes will own great potential in applications such as targeted photoacoustic imaging and photothermal therapy in the near future.

  15. Installing amino acids and peptides on N-heterocycles under visible-light assistance.

    PubMed

    Jin, Yunhe; Jiang, Min; Wang, Hui; Fu, Hua

    2016-02-02

    Readily available natural α-amino acids are one of nature's most attractive and versatile building blocks in synthesis of natural products and biomolecules. Peptides and N-heterocycles exhibit various biological and pharmaceutical functions. Conjugation of amino acids or peptides with N-heterocycles provides boundless potentiality for screening and discovery of diverse biologically active molecules. However, it is a great challenge to install amino acids or peptides on N-heterocycles through formation of carbon-carbon bonds under mild conditions. In this article, eighteen N-protected α-amino acids and three peptides were well assembled on phenanthridine derivatives via couplings of N-protected α-amino acid and peptide active esters with substituted 2-isocyanobiphenyls at room temperature under visible-light assistance. Furthermore, N-Boc-proline residue was successfully conjugated with oxindole derivatives using similar procedures. The simple protocol, mild reaction conditions, fast reaction, and high efficiency of this method make it an important strategy for synthesis of diverse molecules containing amino acid and peptide fragments.

  16. Installing amino acids and peptides on N-heterocycles under visible-light assistance

    PubMed Central

    Jin, Yunhe; Jiang, Min; Wang, Hui; Fu, Hua

    2016-01-01

    Readily available natural α-amino acids are one of nature’s most attractive and versatile building blocks in synthesis of natural products and biomolecules. Peptides and N-heterocycles exhibit various biological and pharmaceutical functions. Conjugation of amino acids or peptides with N-heterocycles provides boundless potentiality for screening and discovery of diverse biologically active molecules. However, it is a great challenge to install amino acids or peptides on N-heterocycles through formation of carbon-carbon bonds under mild conditions. In this article, eighteen N-protected α-amino acids and three peptides were well assembled on phenanthridine derivatives via couplings of N-protected α-amino acid and peptide active esters with substituted 2-isocyanobiphenyls at room temperature under visible-light assistance. Furthermore, N-Boc-proline residue was successfully conjugated with oxindole derivatives using similar procedures. The simple protocol, mild reaction conditions, fast reaction, and high efficiency of this method make it an important strategy for synthesis of diverse molecules containing amino acid and peptide fragments. PMID:26830014

  17. Tumor-penetration and antitumor efficacy of cetuximab are enhanced by co-administered iRGD in a murine model of human NSCLC

    PubMed Central

    Zhang, Yang; Yang, Jie; Ding, Manhua; Li, Liantao; Lu, Zheng; Zhang, Qing; Zheng, Junnian

    2016-01-01

    Lung cancer is the leading cause of cancer-associated mortality, worldwide. For this reason, novel therapies are required for the treatment of this devastating disease. Cetuximab is a monoclonal antibody against epidermal growth factor receptor (EGFR), which is overexpressed in a variety of solid tumors, including non-small cell lung cancer (NSCLC). The therapeutic efficacy of cetuximab for NSCLC is limited to use as a monotherapy or in combination with chemotherapy. The objective of the present study was to develop a novel strategy to enhance the therapeutic efficacy of cetuximab for NSCLC by a co-administration with the tumor-penetrating internalizing RGD peptide (iRGD). Human NSCLC subcutaneous xenograft models established with the A549 cell line in nude mice were treated with 30 mg/kg cetuximab, 4 mg/kg iRGD, cetuximab plus iRGD or phosphate-buffered saline. The tumor-penetration, in vivo therapeutic efficacy and involved mechanism were evaluated. The present study showed that the A549 xenograft model is sensitive to the co-administration of cetuximab and iRGD. Treatment with cetuximab plus iRGD resulted in a significant increase in the tumor-penetration of cetuximab and tumor reduction compared with cetuximab monotherapy. In conclusion, iRGD enhances the effects of co-administered cetuximab in an NSCLC model. The combined application of cetuximab and iRGD may be a novel strategy to enhance the clinical therapeutic efficacy of cetuximab for the treatment of NSCLC. PMID:27899989

  18. Formation of Amino Acid Thioesters for Prebiotic Peptide Synthesis: Catalysis By Amino Acid Products

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.; DeVincenzi, Donald L. (Technical Monitor)

    1999-01-01

    The origin of life can be described as a series of events in which a prebiotic chemical process came increasingly under the control of its catalytic products. In our search for this prebiotic process that yielded catalytic takeover products (such as polypeptides), we have been investigating a reaction system that generates peptide-forming amino acid thioesters from formaldehyde, glycolaldehyde, and ammonia in the presence of thiols. As shown below, this model process begins by aldol condensation of formaldehyde and glycolaldehyde to give trioses and releases. These sugars then undergo beta-dehydration yielding their respective alpha-ketoaldehydes. Addition of ammonia to the alpha-ketoaldehydes yields imines which can either: (a) rearrange in the presence of thesis to give amino acid thioesters or (be react with another molecule of aldehyde to give imidazoles. This 'one-pot' reaction system operates under mild aqueous conditions, and like modem amino acid biosynthesis, uses sugar intermediates which are converted to products by energy-yielding redox reactions. Recently, we discovered that amino acids, such as the alanine reaction product, catalyze the first and second steps of the process. In the presence of ammonia the process also generates other synthetically useful products, like the important biochemical -- pyruvic acid.

  19. Molecular mechanics and dynamics studies on the interaction of gallic acid with collagen-like peptides

    NASA Astrophysics Data System (ADS)

    Madhan, B.; Thanikaivelan, P.; Subramanian, V.; Raghava Rao, J.; Unni Nair, Balachandran; Ramasami, T.

    2001-10-01

    Molecular modelling approaches have been used to understand the interaction of collagen-like peptides with gallic acid, which mimic vegetable tanning processes involved in protein stabilization. Several interaction sites have been identified and the binding energies of the complexes have been calculated. The calculated binding energies for various geometries are in the range 6-13 kcal/mol. It is found that some complexes exhibit hydrogen bonding, and electrostatic interaction plays a dominant role in the stabilization of the peptide by gallic acid. The π-OH type of interaction is also observed in the peptide stabilization. Molecular dynamics (MD) simulation for 600 ps revealed the possibility of hydrogen bonding between the collagen-like peptide and gallic acid.

  20. Noninvasive molecular imaging of MYC mRNA expression in human breast cancer xenografts with a [99mTc]peptide-peptide nucleic acid-peptide chimera.

    PubMed

    Tian, Xiaobing; Aruva, Mohan R; Qin, Wenyi; Zhu, Weizhu; Sauter, Edward R; Thakur, Mathew L; Wickstrom, Eric

    2005-01-01

    Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptide on the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, as well as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenografts scintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissected tissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administration of the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additional genetic characterization of preinvasive and invasive breast cancers.

  1. Activation of carboxyl group with cyanate: peptide bond formation from dicarboxylic acids.

    PubMed

    Danger, Grégoire; Charlot, Solenne; Boiteau, Laurent; Pascal, Robert

    2012-06-01

    The reaction of cyanate with C-terminal carboxyl groups of peptides in aqueous solution was considered as a potential pathway for the abiotic formation of peptide bonds under the condition of the primitive Earth. The catalytic effect of dicarboxylic acids on cyanate hydrolysis was definitely attributed to intramolecular nucleophilic catalysis by the observation of the 1H-NMR signal of succinic anhydride when reacting succinic acid with KOCN in aqueous solution (pH 2.2-5.5). The formation of amide bonds was noticed when adding amino acids or amino acid derivatives into the solution. The reaction of N-acyl aspartic acid derivatives was observed to proceed similarly and the scope of the cyanate-promoted reaction was analyzed from the standpoint of prebiotic peptide formation. The role of cyanate in activating peptide C-terminus constitutes a proof of principle that intramolecular reactions of adducts of peptides C-terminal carboxyl groups with activating agents represent a pathway for peptide activation in aqueous solution, the relevance of which is discussed in connexion with the issue of the emergence of homochirality.

  2. Amino Acid- vs. Peptide-Odorants: Responses of Individual Olfactory Receptor Neurons in an Aquatic Species

    PubMed Central

    Hassenklöver, Thomas; Pallesen, Lars P.; Schild, Detlev; Manzini, Ivan

    2012-01-01

    Amino acids are widely used waterborne olfactory stimuli proposed to serve as cues in the search for food. In natural waters the main source of amino acids is the decomposition of proteins. But this process also produces a variety of small peptides as intermediate cleavage products. In the present study we tested whether amino acids actually are the natural and adequate stimuli for the olfactory receptors they bind to. Alternatively, these olfactory receptors could be peptide receptors which also bind amino acids though at lower affinity. Employing calcium imaging in acute slices of the main olfactory epithelium of the fully aquatic larvae of Xenopus laevis we show that amino acids, and not peptides, are more effective waterborne odorants. PMID:23300867

  3. Synthesis and biological properties of amino acids and peptides containing a tetrazolyl moiety

    NASA Astrophysics Data System (ADS)

    Popova, E. A.; Trifonov, R. E.

    2015-09-01

    Literature data published mainly in the last 15 years on the synthesis and biological properties of amino acid analogues and derivatives containing tetrazolyl moieties are analyzed. Tetrazolyl analogues and derivatives of amino acids and peptides are shown to be promising for medicinal chemistry. Being polynitrogen heterocyclic systems comprising four endocyclic nitrogen atoms, tetrazoles can behave as acids and bases and form strong hydrogen bonds with proton donors (more rarely, with acceptors). They have high metabolic stability and are able to penetrate biological membranes. The review also considers the synthesis and properties of linear and cyclic peptides based on modified amino acids incorporating a tetrazolyl moiety. A special issue is the discussion of the biological properties of tetrazole-containing amino acids and peptides, which exhibit high biological activity and can be used to design new drugs. The bibliography includes 200 references.

  4. A toy model of prebiotic peptide evolution: the possible role of relative amino acid abundances.

    PubMed

    Polanco, Carlos; Buhse, Thomas; Samaniego, José Lino; Castañón González, Jorge Alberto

    2013-01-01

    This paper presents a mathematical-computational toy model based on the assumed dynamic principles of prebiotic peptide evolution. Starting from a pool of amino acid monomers, the model describes in a generalized manner the generation of peptides and their sequential information. The model integrates the intrinsic and dynamic key elements of the initiation of biopolymerization, such as the relative amino acid abundances and polarities, as well as the oligomer reversibility, i.e. fragmentation and recombination, and peptide self-replication. Our modeling results suggest that the relative amino acid abundances, as indicated by Miller-Urey type electric discharge experiments, played a principal role in the early sequential information of peptide profiles. Moreover, the computed profiles display an astonishing similarity to peptide profiles observed in so-called biological common ancestors found in the following three microorganisms; E. coli, M. jannaschii, and S. cereviasiae. The prebiotic peptide fingerprint was obtained by the so-called polarity index method that was earlier reported as a tool for the identification of cationic amphipathic antibacterial short peptides.

  5. Analysis of Endogenous D-Amino Acid-Containing Peptides in Metazoa

    PubMed Central

    Bai, Lu; Sheeley, Sarah; Sweedler, Jonathan V.

    2010-01-01

    Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use L-amino acids to make proteins, a group of D-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the D-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-L sequence. Because this L-to-D post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides. PMID:20490347

  6. The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

    NASA Technical Reports Server (NTRS)

    Nelson, Kevin E.; Miller, Stanley L.

    1992-01-01

    The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or

  7. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  8. Peptide nucleic acid (PNA): a model structure for the primordial genetic material?

    PubMed

    Nielsen, P E

    1993-12-01

    It is proposed that the primordial genetic material could have been peptide nucleic acids, i.e., DNA analogues having a peptide backbone. PNA monomers based on the amino acid, alpha, gamma-diaminobutyric acid or ornithine are suggested as compounds that could have been formed in the prebiotic soup. Finally, the possibility of a PNA/RNA world is presented, in which PNA constitutes the stable genetic material, while RNA which may be polymerized using the PNA as template accounts for enzymatic activities including PNA replication.

  9. The enthalpies of formation and sublimation of amino acids and peptides

    NASA Astrophysics Data System (ADS)

    Sagadeev, E. V.; Gimadeev, A. A.; Barabanov, V. P.

    2010-02-01

    The experimental enthalpies of formation of L-amino acids and peptides were analyzed using the additive scheme and group contributions. Group contributions to the enthalpies of formation were calculated (increment denotations corresponded to the Benson-Buss symbols). The thermochemical characteristics of a wide range of amino acids and their derivatives were calculated.

  10. Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

    PubMed Central

    2013-01-01

    Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues. PMID:24328107

  11. MAGP2 controls Notch via interactions with RGD binding integrins: Identification of a novel ECM-integrin-Notch signaling axis.

    PubMed

    Deford, Peter; Brown, Kasey; Richards, Rae Lee; King, Aric; Newburn, Kristin; Westover, Katherine; Albig, Allan R

    2016-02-01

    Canonical Notch signaling involves Notch receptor activation via interaction with cell surface bound Notch ligand. Recent findings also indicate that Notch signaling may be modulated by cross-talk with other signaling mechanisms. The ECM protein MAGP2 was previously shown to regulate Notch in a cell type dependent manner, although the molecular details of this interaction have not been dissected. Here, we report that MAGP2 cell type specific control of Notch is independent of individual Notch receptor-ligand combinations but dependent on interaction with RGD binding integrins. Overexpressed MAGP2 was found to suppress transcriptional activity from the Notch responsive Hes1 promoter activity in endothelial cells, while overexpression of a RGD→RGE MAGP2 mutant increased Notch signaling in the same cell type. This effect was not unique to MAGP2 since the RGD domain of the ECM protein EGFL7 was also found to be an important modulator of Hes1 promoter activity. Independently of MAGP2 or EGFL7, inhibition of RGD-binding integrins with soluble RGD peptides also increased accumulation of active N1ICD fragments and Notch responsive promoter activity independently of changes in Notch1, Jag1, or Dll4 expression. Finally, β1 or β3 integrin blocking antibodies also enhanced Notch signaling. Collectively, these results answer the question of how MAGP2 controls cell type dependent Notch signaling, but more importantly uncover a new mechanism to understand how extracellular matrices and cellular environments impact Notch signaling.

  12. Predicting three-dimensional conformations of peptides constructed of only glycine, alanine, aspartic acid, and valine.

    PubMed

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  13. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    NASA Astrophysics Data System (ADS)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  14. REACTION OF AMINO-ACIDS AND PEPTIDE BONDS WITH FORMALDEHYDE AS MEASURED BY CHANGES IN THE ULTRA-VIOLET SPECTRA,

    DTIC Science & Technology

    AMINO ACIDS , CHEMICAL REACTIONS), (*PEPTIDES, CHEMICAL REACTIONS), (*FORMALDEHYDE, CHEMICAL REACTIONS), (*ULTRAVIOLET SPECTROSCOPY, PROTEINS), ABSORPTION SPECTRA, CHEMICAL BONDS, AMIDES, CHEMICAL EQUILIBRIUM, REACTION KINETICS

  15. Peptide modules for overcoming barriers of nucleic acids transport to cells.

    PubMed

    Egorova, Anna A; Kiselev, Anton V

    2016-01-01

    Absence of safe and efficient methods of nucleic acids delivery is one of the major issues which limits the development of human gene therapy. Highly efficient viral vectors raise questions due to safety reasons. Among non-viral vectors peptide-based carriers can be considered as good candidates for the development of "artificial viruses"--multifunctional polyplexes that mimic viruses. Suggested strategy to obtain multifunctionality is to combine several peptide modules into one modular carrier. Different kinds of peptide modules are needed for successful overcoming barriers of nucleic acids transport into the cells. Design of such modules and establishment of structure-function relationships are issues of importance to researchers working in the field of nucleic acids delivery.

  16. Effect of Fatty Acid Conjugation on Antimicrobial Peptide Activity

    DTIC Science & Technology

    2004-12-01

    killing mechanism of antimicrobial peptides makes them an interesting alternative to traditional antibiotics, as target bacteria may be less able...C14-AKK and C16-AKK to within a 7% error are 220 and 16mM respectively. Since amphipathicity is requisite for antimicrobial action KAK is not...Schnaare, 2000: Antimicrobial evaluation of N-alkyl betaines and N-alkyl-N,N-dimethylamine oxides with variations in chain length. Antimicrobial Agents

  17. Targeting pre-miRNA by Peptide Nucleic Acids

    PubMed Central

    Avitabile, Concetta; Saviano, Michele; D'Andrea, Luca; Bianchi, Nicoletta; Fabbri, Enrica; Brognara, Eleonora; Gambari, Roberto; Romanelli, Alessandra

    2012-01-01

    PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs. PMID:22699795

  18. Effects of Acidic Peptide Size and Sequence on Trivalent Praseodymium Adduction and Electron Transfer Dissociation Mass Spectrometry.

    PubMed

    Commodore, Juliette J; Cassady, Carolyn J

    2017-02-07

    Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr - H](2+) ; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues, generate [M + Pr](3+) , which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H](4+) ; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non-metallated c- and z-ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N-terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal-peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III).

  19. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability.

    PubMed

    Tang, Ning; Skibsted, Leif H

    2016-06-01

    Deprotonation of amino acids as occurs during transfer from stomach to intestines during food digestion was found by comparison of complex formation constants as determined electrochemically for increasing pH to increase calcium binding (i) by a factor of around 6 for the neutral amino acids, (ii) by a factor of around 4 for anions of the acidic amino acids aspartic and glutamic acid, and (iii) by a factor of around 5.5 for basic amino acids. Optimized structures of the 1:1 complexes and ΔHbinding for calcium binding as calculated by density functional theory (DFT) confirmed in all complexes a stronger calcium binding and shorter calcium-oxygen bond length in the deprotonated form. In addition, the stronger calcium binding was also accompanied by a binding site shift from carboxylate binding to chelation by α-amino group and carboxylate oxygen for leucine, aspartate, glutamate, alanine, and asparagine. For binary amino acid mixtures, the calcium-binding constant was close to the predicted geometric mean of the individual amino acid binding constants indicating separate binding of calcium to two amino acids when present together in solution. At high pH, corresponding to conditions for calcium absorption, the binding affinity increased in the order Lys < Arg < Cys < Gln < Gly ∼ Ala < Asn < His < Leu < Glu< Asp. In a series of glycine peptides, calcium-binding affinity was found to increase in the order Gly-Leu ∼ Gly-Gly < Ala-Gly < Gly-His ∼ Gly-Lys-Gly < Glu-Cys-Gly < Gly-Glu, an ordering confirmed by DFT calculations for the dipeptides and which also accounted for large synergistic effects in calcium binding for up to 6 kJ/mol when compared to the corresponding amino acid mixtures.

  20. Predicting anticancer peptides with Chou's pseudo amino acid composition and investigating their mutagenicity via Ames test.

    PubMed

    Hajisharifi, Zohre; Piryaiee, Moien; Mohammad Beigi, Majid; Behbahani, Mandana; Mohabatkar, Hassan

    2014-01-21

    Cancer is an important reason of death worldwide. Traditional cytotoxic therapies, such as radiation and chemotherapy, are expensive and cause severe side effects. Currently, design of anticancer peptides is a more effective way for cancer treatment. So there is a need to develop a computational method for predicting the anticancer peptides. In the present study, two methods have been developed to predict these peptides using support vector machine (SVM) as a powerful machine learning algorithm. Classifiers have been applied based on the concept of Chou's pseudo-amino acid composition (PseAAC) and local alignment kernel. Since a number of HIV-1 proteins have cytotoxic effect, therefore we predicted the anticancer effect of HIV-1 p24 protein with these methods. After the prediction, mutagenicity of 2 anticancer peptides and 2 non-anticancer peptides was investigated by Ames test. Our results show that, the accuracy and the specificity of local alignment kernel based method are 89.7% and 92.68%, respectively. The accuracy and specificity of PseAAC-based method are 83.82% and 85.36%, respectively. By computational analysis, out of 22 peptides of p24 protein, 4 peptides are anticancer and 18 are non-anticancer. In the Ames test results, it is clear that anticancer peptides (ARP788.8 and ARP788.21) are not mutagenic. Therefore the results demonstrate that the described computation methods are useful to identify potential anticancer peptides, which are worthy of further experimental validation and 2 peptides (ARP788.8 and ARP788.21) of HIV-1 p24 protein can be used as new anticancer candidates without mutagenicity.

  1. A Tumor-Penetrating Peptide Modification Enhances the Antitumor Activity of Thymosin Alpha 1

    PubMed Central

    Chen, Jiao; Zheng, Heng

    2013-01-01

    A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment. PMID:23977262

  2. A tumor-penetrating peptide modification enhances the antitumor activity of thymosin alpha 1.

    PubMed

    Lao, Xingzhen; Liu, Meng; Chen, Jiao; Zheng, Heng

    2013-01-01

    A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment.

  3. Peptide nucleic acid (PNA): A model structure for the primordial genetic material?

    NASA Astrophysics Data System (ADS)

    Nielsen, Peter Egil

    1993-12-01

    It is proposed that the primordial genetic material could have been peptide nucleic aicds,i.e., DNA analogues having a peptide backbone. PNA momomers based on the amino acid, α, γ-diaminobutyric acid or ornithine are suggested as compounds that could have been formed in the prebiotic soup. Finally, the possibility of a PNA/RNA world is presented, in which PNA constitutes the stable genetic material, while RNA which may be polymerized using the PNA as template accounts for enzymatic activities including PNA replication.

  4. [Antiaggregation activity of arachidonic acid conjugates with neurotropic peptides proglyprol and semax].

    PubMed

    Bezuglov, V V; Gretskaia, N M; Vasil'eva, T M; Petrukhina, G N; Andreeva, L A; Miasoedov, N F; Makarov, V A

    2014-01-01

    The influence two original derivatives of a therapeutically important peptide, bearing arachidonic acid residue with semax and proglyprol, upon platelet aggregation have been studied in vitro. It is established that both derivatives, in contrast to the parent peptide, possess moderate anti-aggregant properties and produce a dose-dependent decrease in the interplatelet interaction induced by ADP, epinephrine, and arachidonic acid within the concentration range of 0.018 - 1.8 mM. This activity was more pronounced for arachidonoylsemax in comparison with arachidonoylproglyprol.

  5. Monocyte activation in response to polyethylene glycol hydrogels grafted with RGD and PHSRN separated by interpositional spacers of various lengths.

    PubMed

    Schmidt, David Richard; Kao, Weiyuan John

    2007-12-01

    Polyethylene glycol (PEG) is often cited as a "stealth" polymer, capable of resisting both protein adsorption and cell adhesion. By extension, PEG would then be expected to limit the host response. Monocyte-derived macrophages play an integral role in inflammation, and thus their response to a material can potentially dictate the overall host response to a biomaterial. In the present study, monocyte responses following interaction with a photopolymerized PEG hydrogel were compared with those from standard tissue culture polystyrene (TCPS). Additionally, the effect of the spacing between RGD and PHSRN, the corresponding synergy sequence on fibronectin (FN), was evaluated using peptides with differing spacer lengths grafted to the PEG hydrogel. Monocyte adherent density on the PEG-only hydrogel was comparable with that of TCPS; however, the secretion of the proinflammatory molecules interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) increased dramatically following monocyte interaction with PEG-only hydrogels as compared with TCPS. The matrix metalloproteinase-2 (MMP-2) concentration was similar for all surfaces, while both the matrix metalloproteinase-9 (MMP-9) and FN concentrations were above the range of the assay for all substrates. Cell density was higher on the PHSRNG(13)RGD grafted substrate as compared with PHSRNG(6)RGD, but neither sequence increased cell density versus RGD alone. Although protein concentration did sometimes vary with different peptides, this variation was minimal in comparison with the surface effects between TCPS and the PEG-only hydrogel. This study explores the roles of PEG and FN-derived peptides on monocyte activation.

  6. Synthesis of lipoic acid-peptide conjugates and their effect on collagen and melanogenesis.

    PubMed

    Lu, Chichong; Kim, Bo Mi; Lee, Duckhee; Lee, Min Hee; Kim, Jin Hwa; Pyo, Hyeong-Bae; Chai, Kyu Yun

    2013-11-01

    We report new examples of lipoic acid (LA)-peptide conjugates, their potential as codrugs having anti-melanogenic and anti-aging properties was evaluated. These multifunctional molecules were prepared by linking lipophilic moiety (LA) to the pentapeptide KTTKS. The inhibitory effect of LA-peptide conjugates on melanin synthesis and tyrosinase activity is stronger than that of LA or the pentapeptide alone. Importantly, the conjugates display no cytotoxicity at a high concentration. LA-KTTKS and LA-PEG-KTTKS also inhibit UV-induced matrix metalloproteinase-1 expression up to 49.5% and 69.5% at 0.5 mM, respectively. LA-peptide conjugates stimulate collagen biosynthesis in fibroblasts more efficiently than their parent molecules do. These data suggest that LA-peptide conjugates may have cosmeceutical application as anti-melanogenic and anti-aging agents.

  7. Surface Functionalization of Piezoelectric Aluminum Nitride with Selected Amino Acid and Peptides

    NASA Astrophysics Data System (ADS)

    Chan, Edmund Ho Man

    In the present contribution, we elaborate on the covalent attachment of the amino acid cysteine and selected cysteine-bearing peptides, in aqueous buffered media, onto AlN surfaces modified with adlayers of one of our homemade bifunctional alkyltrichlorosilane cross-linking molecules bearing the benzenethiosulfonate head group. Surface characterizations confirmed the successful covalent immobilization of cysteine in buffered media, whereas the attachment of the peptides proved to be difficult as the undesired partial destruction of the adlayer on AlN by hydrolysis in aqueous/buffered solvent systems, which was confirmed in a separate study, appeared to have interfered with the covalent attachment and resulted in one of the peptides failing to immobilize. Future directions from this will focus on optimizing the solvent conditions for the cysteine/peptide immobilizations and the implementation of the surface chemistry to the covalent functionalization of AlN with biologically significant protein fragments, among them the antigen-binding fragment of antibodies.

  8. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis

    NASA Astrophysics Data System (ADS)

    Osberger, Thomas J.; Rogness, Donald C.; Kohrt, Jeffrey T.; Stepan, Antonia F.; White, M. Christina

    2016-09-01

    Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C-H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C-H oxidation strategy is demonstrated in its capacity for generating diversity: four ‘chiral pool’ amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C-H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C-H oxidation to one containing a linear unnatural amino acid.

  9. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis.

    PubMed

    Osberger, Thomas J; Rogness, Donald C; Kohrt, Jeffrey T; Stepan, Antonia F; White, M Christina

    2016-09-08

    Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C-H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C-H oxidation strategy is demonstrated in its capacity for generating diversity: four 'chiral pool' amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C-H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C-H oxidation to one containing a linear unnatural amino acid.

  10. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    PubMed

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  11. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  12. Antimicrobial peptides incorporating non-natural amino acids as agents for plant protection.

    PubMed

    Ng-Choi, Iteng; Soler, Marta; Güell, Imma; Badosa, Esther; Cabrefiga, Jordi; Bardaji, Eduard; Montesinos, Emilio; Planas, Marta; Feliu, Lidia

    2014-04-01

    The control of plant pathogens is mainly based on copper compounds and antibiotics. However, the use of these compounds has some limitations. They have a high environmental impact and the use of antibiotics is not allowed in several countries. Moreover, resistance has been developed to these pathogens. The identification of new agents able to fight plant pathogenic bacteria and fungi will represent an alternative to currently used antibiotics or pesticides. Antimicrobial peptides are widely recognized as promising candidates, however naturally occurring sequences present drawbacks that limit their development. These include susceptibility to protease degradation and low bioavailability. To overcome these problems, research has focused on the introduction of unnatural amino acids into lead peptide sequences. In particular, we have improved the biological profile of antimicrobial peptides active against plant pathogenic bacteria and fungi by incorporating triazolyl, biaryl and D-amino acids into their sequence. These modifications and their influence on the biological activity are summarized.

  13. Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

    PubMed

    Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

    2017-03-01

    Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

  14. Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

    NASA Astrophysics Data System (ADS)

    DeGraan-Weber, Nick; Zhang, Jun; Reilly, James P.

    2016-12-01

    Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues.

  15. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage.

    PubMed

    Barlos, K; Chatzi, O; Gatos, D; Stavropoulos, G

    1991-06-01

    The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr.

  16. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin

    PubMed Central

    Makartsova, Anna A.; Fomin, Alexandr S.; Nushtaeva, Anna A.; Koval, Olga A.

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  17. Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

    NASA Astrophysics Data System (ADS)

    McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

    2016-05-01

    Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

  18. Selected Lactic Acid Bacteria Synthesize Antioxidant Peptides during Sourdough Fermentation of Cereal Flours

    PubMed Central

    Coda, Rossana; Pinto, Daniela; Gobbetti, Marco

    2012-01-01

    A pool of selected lactic acid bacteria was used for the sourdough fermentation of various cereal flours with the aim of synthesizing antioxidant peptides. The radical-scavenging activity of water/salt-soluble extracts (WSE) from sourdoughs was significantly (P < 0.05) higher than that of chemically acidified doughs. The highest activity was found for whole wheat, spelt, rye, and kamut sourdoughs. Almost the same results were found for the inhibition of linoleic acid autoxidation. WSE were subjected to reverse-phase fast protein liquid chromatography. Thirty-seven fractions were collected and assayed in vitro. The most active fractions were resistant to further hydrolysis by digestive enzymes. Twenty-five peptides of 8 to 57 amino acid residues were identified by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. Almost all of the sequences shared compositional features which are typical of antioxidant peptides. All of the purified fractions showed ex vivo antioxidant activity on mouse fibroblasts artificially subjected to oxidative stress. This study demonstrates the capacity of sourdough lactic acid bacteria to release peptides with antioxidant activity through the proteolysis of native cereal proteins. PMID:22156436

  19. Novel Bifunctional Cyclic Chelator for 89Zr Labeling–Radiolabeling and Targeting Properties of RGD Conjugates

    PubMed Central

    2015-01-01

    Within the last years 89Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting αvß3 integrins were synthesized using different conjugation strategies and labeled with 89Zr. In vitro stability, biodistribution, and microPET/CT imaging were evaluated using [89Zr]FSC-RGD conjugates or [89Zr]triacetylfusarinine C (TAFC). Quantitative 89Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [89Zr]DFO, [89Zr]FSC derivatives showed excellent in vitro stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [89Zr]FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high in vivo stability. These findings indicate that FSC is a highly promising chelator for the development of 89Zr-based PET imaging agents. PMID:25941834

  20. Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor.

    PubMed

    Sant'Angelo, Derek B; Robinson, Eve; Janeway, Charles A; Denzin, Lisa K

    2002-09-01

    CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.

  1. RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

    PubMed Central

    1990-01-01

    Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with

  2. Laser ion beam photodissociation studies of model amino acids and peptides

    SciTech Connect

    Techlenburg, R.E. Jr.; Miller, M.N.; Russell, D.H. )

    1989-02-15

    Visible (458-514.5 nm) and uv (333-385 nm) photodissociation of the (M + H){sup +} ions of dinitrophenyl (DNP) derivatized amino acids and peptides is reported. Photoexcitation of the DNP peptides by a visible proton results in fragmentation of the peptide chain with little fragmentation within the chromophore. Conversely, uv photoexcitation of the DNP peptides results in fragmentation of the chromophore as well as the peptide chain, but loss of NO or NO{sub 2} (within the chromophore) often dominates the photofragment ion spectrum. These results are rationalized with particular emphasis on energy-selective dissociation channels of large ionic systems. DNP-leucine and DNP-isoleucine (M + H){sup +} can be differentiated on the basis of photodissociation reactions which yield distonic radical cations. The rate of dissociation of photoexcited ions of DNP peptides is shown to decrease with increasing molecular weight (degrees of freedom). Lastly, comparisons between photodissociation and collision-induced dissociation as a structural probe are presented. 55 refs., 8 figs., 3 tabs.

  3. Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays.

    PubMed

    Weber, Laura K; Palermo, Andrea; Kügler, Jonas; Armant, Olivier; Isse, Awale; Rentschler, Simone; Jaenisch, Thomas; Hubbuch, Jürgen; Dübel, Stefan; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F

    2017-04-01

    The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.

  4. Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

    PubMed Central

    Volzing, Katherine; Borrero, Juan; Sadowsky, Michael J.; Kaznessis, Yiannis N.

    2014-01-01

    We present results of tests with recombinant Lactococcus lactis that produce and secrete heterologous antimicrobial peptides with activity against Gram-negative pathogenic Escherichia coli and Salmonella. In an initial screening, the activities of numerous candidate antimicrobial peptides, made by solid state synthesis, were assessed against several indicator pathogenic E. coli and Salmonella strains. Peptides A3APO and Alyteserin were selected as top performers based on high antimicrobial activity against the pathogens tested and on significantly lower antimicrobial activity against L. lactis. Expression cassettes containing the signal peptide of the protein Usp45 fused to the codon optimized sequence of mature A3APO and Alyteserin were cloned under the control of a nisin-inducible promoter nisA and transformed into L. lactis IL1403. The resulting recombinant strains were induced to express and secrete both peptides. A3APO- and Alyteserin-containing supernatants from these recombinant L. lactis inhibited the growth of pathogenic E. coli and Salmonella by up to 20-fold, while maintaining the host’s viability. This system may serve as a model for the production and delivery of antimicrobial peptides by lactic acid bacteria to target Gram-negative pathogenic bacteria populations. PMID:23808914

  5. HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma.

    PubMed

    Martellini, Julie A; Cole, Amy L; Svoboda, Pavel; Stuchlik, Olga; Chen, Li-Mei; Chai, Karl X; Gangrade, Bhushan K; Sørensen, Ole E; Pohl, Jan; Cole, Alexander M

    2011-01-20

    We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

  6. Aromatic amino acids providing characteristic motifs in the Raman and SERS spectroscopy of peptides.

    PubMed

    Wei, Fang; Zhang, Dongmao; Halas, Naomi J; Hartgerink, Jeffrey D

    2008-07-31

    Raman and surface-enhanced Raman spectroscopies (SERS) are potentially important tools in the characterization of biomolecules such as proteins and DNA. In this work, SERS spectra of three cysteine-containing aromatic peptides: tryptophan-cysteine, tyrosine-cysteine, and phenylalanine-cysteine, bound to Au nanoshell substrates, were obtained, and compared to their respective normal Raman spectra. While the linewidths of the SERS peaks are significantly broadened (up to 70%), no significant spectral shifts (<6 cm (-1)) of the major Stokes modes were observed between the two modalities. We show that the Raman and SERS spectra of penetratin, a cell-penetrating peptide oligomer, can be comprised quite reliably from the spectra of its constituent aromatic amino acids except in the backbone regions where the spectral intensities are critically dependent on the length and conformations of the probed molecules. From this study we conclude that, together with protein backbone groups, aromatic amino acid residues provide the overwhelmingly dominant features in the Raman and SERS spectra of peptides and proteins when present. It follows that the Raman modes of these three small constructed peptides may likely apply to the assignment of Raman and SERS features in the spectra of other peptides and proteins.

  7. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis

    PubMed Central

    Osberger, Thomas J.; Rogness, Donald C.; Kohrt, Jeffrey T.; Stepan, Antonia F.; White, M. Christina

    2016-01-01

    Secondary metabolites synthesized by nonribosomal peptide synthetases (NRPSs) display diverse and complex topologies and possess an impressive range of biological activities1,2 Much of this diversity derives from a synthetic strategy that entails the oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds pre-3 and post-assembly2. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis.4 However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized outside of nature.5 In this manuscript, we report that two small-molecule iron catalysts are capable of facilitating the targeted C—H oxidative modification of amino acids and peptides with preservation of α-center chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins, and amines in both monomer and peptide settings. The value of this C—H oxidation strategy is demonstrated in its capacity for generating diversity: four 'chiral pool' amino acids are transformed to twenty-one chiral unnatural amino acids (UAAs) representing seven distinct functional group arrays; late-stage C—H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different UAAs. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C—H oxidation to one containing a linear UAA. PMID:27479323

  8. Deoxycholate Bile Acid Directed Synthesis of Branched Au Nanostructures for Near Infrared Photothermal Ablation

    PubMed Central

    Kim, Dong-Hyun; Larson, Andrew C.

    2015-01-01

    We report an approach for simple, reproducible and high-yield synthesis of branched GNPs directed by deoxycholate bile acid supramolecular aggregates in Au solution. A growth process involving stepwise trapping of the GNP seeds and Au ions in the deoxycholate bile acid solution yields multiple-branched GNPs. Upon NIR laser irradiation strong NIR absorption for branched GNPs induced photothermal-heating to destroy tumor cells. Subsequently, these branched GNPs were bio-functionalized with cRGD cell penetrating-targeting peptides for photothermal cancer treatment applications. Branched GNPs conjugated with cRGD peptides enhanced internalization of the branched GNPs in BxPC3 human pancreatic adenocarcinoma cells and effectively ablated BxPC3 cells when irradiated with a NIR laser (808 nm). Their potential use as photothermal transducing agents was demonstrated in in vivo settings using a pancreatic cancer xenograft model. The tumors were effectively ablated with cRGD-branched GNPs injection and laser exposure without any observation of tumor recurrence. This firstly reported method for deoxycholate bile acid directed synthesis of branched GNPs opens new possibilities for the production of strong NIR absorbing nanostructures for selective nanophotothermolisys of cancer cells and the further design of novel materials with customized spectral and structural properties for broader applications. PMID:25934288

  9. Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria.

    PubMed

    Scott, M G; Gold, M R; Hancock, R E

    1999-12-01

    Compounds with antiendotoxin properties have been extensively studied for their potential as therapeutic agents for sepsis attributable to gram-negative bacteria. However, with the increasing incidence of gram-positive sepsis, there is interest in identifying compounds with a broad spectrum of action against both gram-positive and gram-negative bacteria. A series of synthetic alpha-helical cationic peptides related to bee melittin and silk moth cecropin have previously been shown to bind lipopolysaccharide (LPS) with high affinity, inhibit LPS-induced tumor necrosis factor alpha (TNF-alpha) production in vitro and in vivo, and kill gram-negative bacteria. In this study, we analyzed whether these peptides were active against gram-positive bacteria; whether they could bind to lipoteichoic acid (LTA), the major proinflammatory structure on gram-positive bacteria; and whether they could block the ability of LTA to promote the release of cytokines by the RAW 264.7 murine macrophage cell line. We found that the cationic peptides demonstrated moderate growth-inhibitory activity toward gram-positive bacteria. In addition, the peptides bound LTA with high affinity. This correlated with the ability of the peptides to block LTA-induced production of TNF and interleukin-6 by RAW 264.7 cells but did not correlate with their ability to kill the bacteria. The peptides also effectively inhibited LTA-induced TNF production in a whole human blood assay. The peptides were also able to partly block the ability of heat-killed Staphylococcus aureus, as well as soluble products of live S. aureus, to stimulate cytokine production by macrophages. Our results indicate that these cationic peptides may be useful to prevent sepsis and inflammation caused by both gram-negative and gram-positive bacteria.

  10. Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

    PubMed Central

    Marco-Urrea, Ernest; Seifert, Jana; von Bergen, Martin

    2012-01-01

    Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. PMID:22661690

  11. Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

    PubMed

    Schweitzer-Stenner, Reinhard; Toal, Siobhan E

    2014-11-07

    As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

  12. The Unexpected Advantages of Using D-Amino Acids for Peptide Self-Assembly into Nanostructured Hydrogels for Medicine

    PubMed Central

    Melchionna, Michele; Styan, Katie E.; Marchesan, Silvia

    2016-01-01

    Self-assembled peptide hydrogels have brought innovation to the medicinal field, not only as responsive biomaterials but also as nanostructured therapeutic agents or as smart drug delivery systems. D-amino acids are typically introduced to increase the peptide enzymatic stability. However, there are several reports of unexpected effects on peptide conformation, self-assembly behavior, cytotoxicity and even therapeutic activity. This mini-review discusses all the surprising twists of heterochiral self-assembled peptide hydrogels, and delineates emerging key findings to exploit all the benefits of D-amino acids in this novel medicinal area. PMID:26876522

  13. Question 1: Peptide nucleic acids and the origin and homochirality of life.

    PubMed

    Nielsen, Peter E

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  14. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

    PubMed

    Periat, Aurélie; Krull, Ira S; Guillarme, Davy

    2015-02-01

    This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.

  15. Stabilization Effect of Amino Acid Side Chains in Peptide Assemblies on Graphite Studied by Scanning Tunneling Microscopy.

    PubMed

    Guo, Yuanyuan; Hou, Jingfei; Zhang, Xuemei; Yang, Yanlian; Wang, Chen

    2017-02-03

    An analysis is presented of the effects of amino acid side chains on peptide assemblies in ambient conditions on a graphite surface. The molecularly resolved assemblies of binary peptides are examined with scanning tunneling microscopy. A comparative analysis of the assembly structures reveals that the lamellae width has an appreciable dependence on the peptide sequence, which could be considered as a manifestation of a stabilizing effect of side-chain moieties of amino acids with high (phenylalanine) and low (alanine, asparagine, histidine and aspartic acid) propensities for aggregation. These amino acids are representative for the chemical structures involving the side chains of charged (histidine and aspartic acid), aromatic (phenylalanine), hydrophobic (alanine), and hydrophilic (asparagine) amino acids. These results might provide useful insight for understanding the effects of sequence on the assembly of surface-bound peptides.

  16. Pharmacological Characterization of the αvβ6 Integrin Binding and Internalization Kinetics of the Foot-and-Mouth Disease Virus Derived Peptide A20FMDV2.

    PubMed

    Slack, Robert J; Hafeji, Maryam; Rogers, Rebecca; Ludbrook, Steve B; Marshall, John F; Flint, David J; Pyne, Susan; Denyer, Jane C

    2016-01-01

    A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvβ6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvβ6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (K(D): 0.22 nmol/l) and selectivity (at least 85-fold) for αvβ6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvβ6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvβ6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t(1/2): 1.5 and 3.1 min, respectively), concentration-dependent (EC50: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvβ6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvβ6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvβ6 following RGD ligand engagement.

  17. Enhancement of acid tolerance in Zymomonas mobilis by a proton-buffering peptide.

    PubMed

    Baumler, David J; Hung, Kai F; Bose, Jeffrey L; Vykhodets, Boris M; Cheng, Chorng M; Jeong, Kwang-Cheol; Kaspar, Charles W

    2006-07-01

    A portion of the cbpA gene from Escherichia coli K-12 encoding a 24 amino acid proton-buffering peptide (Pbp) was cloned via the shuttle vector pJB99 into E. coli JM105 and subsequently into Zymomonas mobilis CP4. Expression of Pbp was confirmed in both JM105 and CP4 by HPLC. Z. mobilis CP4 carrying pJB99-2 (Pbp) exhibited increased acid tolerance (p < 0.05) in acidified TSB (HCl [pH 3.0] or acetic acid [pH 3.5]), glycine-HCl buffer (pH 3.0), and sodium acetate-acetic acid buffer (pH 3.5) in comparison to the parent strain (CP4) and CP4 with pJB99 (control plasmid). Although the expression of Pbp influenced survival at a low pH, the minimum growth pH was unaffected. Growth of Z. mobilis in the presence of ampicillin also significantly increased acid tolerance by an unknown mechanism. Results from this study demonstrate that the production of a peptide with a high proportion of basic amino acids can contribute to protection from low pH and weak organic acids such as acetic acid.

  18. Mass spectral study of hybrid peptides derived from (R)-aminoxy ester and [beta]-amino acids: The influence of aminoxy peptide bond (CO-NH-O) on peptide fragmentation under electrospray ionization conditions

    NASA Astrophysics Data System (ADS)

    Ramesh, V.; Ramesh, M.; Srinivas, R.; Sharma, G. V. M.; Manohar, V.

    2009-04-01

    A new class of Boc-protected aminoxy hybrid peptides containing repeats of [beta]-hAla-(R)-Ama-, and [beta]-Caa-(R)-Ama- ([beta]-hAla = [beta]3-(S)-hAlanine, (R)-Ama = (R)-aminoxy ester, and [beta]-Caa = (R)-C-linked carbo-[beta]3-amino acid) have been studied by electrospray ionization (ESI) ion-trap and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) of their protonated, cationized, and negative ions. MS3 CID of protonated aminoxy peptides of [beta]-hAla-(R)-Ama- yield intense [beta]-amino acid characteristic retro-Mannich fragmentation. The bn+ and [bn-methyl imine]+ (n = 3, 5) ions formed by cleavage of aminoxy peptide bond (CO-NH-O) are more intense than bn+ (n = 2, 4) formed by that of peptide bond (CO-NH-C) cleavage. Another characteristic ion observed is due to loss of H3NO from yn+ ions. The cationized (Li+, and Na+) peptides dissociate differently compared to protonated peptides. Intense cationized cn and zn ions are formed due to the cleavage of N-O bond. The deprotonated peptides also show abundant cn- and zn- ions (n = 1, 3, 5) and do not form any yn- ions. All these results clearly indicate the influence of aminoxy peptide bond on fragmentation of these hybrid peptides.

  19. [Amino acid and peptide derivatives of the tylosin family of macrolide antibiotics modified at the aldehyde group].

    PubMed

    Sumbatian, N V; Kuznetsova, I V; Karpenko, V V; Fedorova, N V; Chertkov, V A; Korshunova, G A; Bogdanov, A A

    2010-01-01

    Fourteen new functionally active amino acid and peptide derivatives of the antibiotics tylosin, desmycosin, and 5-O-mycaminosyltylonolide were synthesized in order to study the interaction of the growing polypeptide chain with the ribosomal tunnel. The conjugation of various amino acids and peptides with a macrolide aldehyde group was carried out by two methods: direct reductive amination with the isolation of the intermediate Schiff bases or through binding via oxime using the preliminarily obtained derivatives of 2-aminooxyacetic acid.

  20. Properties of synthetic ferrihydrite as an amino acid adsorbent and a promoter of peptide bond formation.

    PubMed

    Matrajt, G; Blanot, D

    2004-03-01

    Ferrihydrite, an iron oxide hydroxide, is found in all kinds of environments, from hydrothermal hot springs to extraterrestrial materials. It has been shown that this material is nanoporous, and because of its high surface area, it has outstanding adsorption properties and in some cases catalysis properties. In this work we studied the adsorption properties of ferrihydrite with respect to amino acids. Samples of pure ferrihydrite were synthesised and exposed to solutions of amino acids including both proteinaceous and non-proteinaceous species. These experiments revealed important characteristics of this mineral as both an adsorbent of amino acids and a promoter of peptide bond formation.

  1. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs.

  2. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins.

  3. Biological Activity of Aminophosphonic Acids and Their Short Peptides

    NASA Astrophysics Data System (ADS)

    Lejczak, Barbara; Kafarski, Pawel

    The biological activity and natural occurrence of the aminophosphonic acids were described half a century ago. Since then the chemistry and biology of this class of compounds have developed into the separate field of phosphorus chemistry. Today it is well acknowledged that these compounds possess a wide variety of promising, and in some cases commercially useful, physiological activities. Thus, they have found applications ranging from agrochemical (with the herbicides glyphosate and bialaphos being the most prominent examples) to medicinal (with the potent antihypertensive fosinopril and antiosteoporetic bisphosphonates being examples).

  4. Role of Amino Acid Insertions on Intermolecular Forces between Arginine Peptide Condensed DNA Helices

    PubMed Central

    DeRouchey, Jason E.; Rau, Donald C.

    2011-01-01

    In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads. PMID:21994948

  5. Lactic Acid Bacteria as Cell Factories for the Generation of Bioactive Peptides.

    PubMed

    Brown, Lucia; Pingitore, Esteban Vera; Mozzi, Fernanda; Saavedra, Lucila; Villegas, Josefina M; Hebert, Elvira M

    2017-01-01

    There is a growing interest in the incorporation of functional foods in the daily diet to achieve health promotion and disease risk reduction. Numerous studies have focused on the production of biologically active peptides as nutraceuticals and functional food ingredients due to their health benefits. These short peptides, displaying antihypertensive, antioxidant, mineral binding, immunomodulatory and antimicrobial activities are hidden in a latent state within the primary sequences of food proteins requiring enzymatic proteolysis for their release. While microbial fermentation is one of the major and economically most convenient processes used to generate bioactive peptides, lactic acid bacteria (LAB) are widely used as starter cultures for the production of diverse fermented foods. This article reviews the current knowledge on LAB as cell factories for the production of bioactive peptides from a variety of food protein sources. These microorganisms depend on a complex proteolytic system to ensure successful fermentation processes. In the dairy industry, LAB containing cell envelope-associated proteinases (CEPs) are employed as biocatalysts for the first step of casein breakdown releasing bioactive peptides during milk fermentation. A better understanding of the functionality and regulation of the proteolytic system of LAB opens up future opportunities for the production of novel food-derived compounds with potential health-promoting properties.

  6. Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.

    PubMed

    Schulze, W; Frommer, W B; Ward, J M

    1999-03-01

    Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.

  7. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  8. Characterisation of neuroprotective efficacy of modified poly-arginine-9 (R9) peptides using a neuronal glutamic acid excitotoxicity model.

    PubMed

    Edwards, Adam B; Anderton, Ryan S; Knuckey, Neville W; Meloni, Bruno P

    2017-02-01

    In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with D-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1-6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.

  9. A tumor-penetrating peptide enhances circulation-independent targeting of peritoneal carcinomatosis

    PubMed Central

    Sugahara, Kazuki N.; Scodeller, Pablo; Braun, Gary B.; de Mendoza, Tatiana Hurtado; Yamazaki, Chisato M.; Kluger, Michael D.; Kitayama, Joji; Alvarez, Edwin; Howell, Stephen B.; Teesalu, Tambet; Ruoslahti, Erkki; Lowy, Andrew M.

    2015-01-01

    Peritoneal carcinomatosis is a major source of morbidity and mortality in patients with advanced abdominal neoplasms. Intraperitoneal chemotherapy (IPC) is an area of intense interest given its efficacy in ovarian cancer. However, IPC suffers from poor drug penetration into peritoneal tumors. As such, extensive cytoreductive surgery is required prior to IPC. Here, we explore the utility of iRGD, a tumor-penetrating peptide, for improved tumor-specific penetration of intraperitoneal compounds and enhanced IPC in mice. Intraperitoneally administered iRGD significantly enhanced penetration of an attached fluorescein into disseminated peritoneal tumor nodules. The penetration was tumor-specific, circulation-independent, and mediated by the neuropilin-binding RXXK tissue-penetration peptide motif of iRGD. Q-iRGD, which fluoresces upon cleavage, including the one that leads to RXXK activation, specifically labeled peritoneal metastases displaying different growth patterns in mice. Importantly, iRGD enhanced intratumoral entry of intraperitoneally co-injected dextran to approximately 300% and doxorubicin to 250%. Intraperitoneal iRGD/doxorubicin combination therapy inhibited the growth of bulky peritoneal tumors and reduced systemic drug toxicity. iRGD delivered attached fluorescein and co-applied nanoparticles deep into fresh human peritoneal metastasis explants. These results indicate that intraperitoneal iRGD co-administration serves as a simple and effective strategy to facilitate tumor detection and improve the therapeutic index of IPC for peritoneal carcinomatosis. PMID:26071630

  10. Systematic amino acid substitutions improved efficiency of GD2-peptide mimotope vaccination against neuroblastoma.

    PubMed

    Bleeke, Matthias; Fest, Stefan; Huebener, Nicole; Landgraf, Christiane; Schraven, Burkhart; Gaedicke, Gerhard; Volkmer, Rudolf; Lode, Holger N

    2009-11-01

    The likelihood of identifying peptides of sufficient quality for the development of effective cancer vaccines by screening of phage display libraries is low. Here, we introduce the sequential application of systematic amino acid substitution by SPOT synthesis. After the substitution of two amino acids within the sequence of a phage display-derived mimotope of disialoganglioside GD2 (mimotope MA), the novel mimotope C3 showed improved GD2 mimicry in vitro. Peptide vaccination with the C3 mimotope induced an 18-fold increased anti-GD2 serum response associated with reduction of primary tumour growth and spontaneous metastasis in contrast to MA mimotope controls in a syngeneic neuroblastoma model. In summary, SPOT provides an ideal optimisation tool for the development of phage display-derived cancer vaccines.

  11. Computer analysis of antigenic domains and RGD-like sequences (RGWG) in the E glycoprotein of flaviviruses: an approach to vaccine development.

    PubMed

    Becker, Y

    1990-09-01

    Antigenic domains and RGD-like sequences in the E glycoprotein of the flaviviruses Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue type 4 virus, and tick-borne encephalitis virus were analyzed by computer programs that provide information on the physical properties of the polypeptides. The use of computer programs for the development of vaccines based on the synthesis of antigenic peptides is discussed. Synthetic viral peptides are proposed to be used for topical application so as to interfere with the virus-cell interaction. Viral peptides with antigenic epitopes to protect against dengue virus infection without enhancing pathogenesis may also be developed on the basis of the computer analysis.

  12. Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Poon, Wilson; Zhang, Xuan; Bekah, Devesh; Teodoro, Jose G.; Nadeau, Jay L.

    2015-07-01

    This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

  13. [Use of peptide bioregulators in intoxication with the herbicide 2,4-dichlorophenoxyacetic acid].

    PubMed

    Lebedeva, S N; Zhamsaranova, S D

    2004-01-01

    The paper shows it promising to use peptide bioregulators--fractions obtained from the cattle immune system (thymus, spleen, and lymph nodes) during immunotherapy for intoxication experimentally caused by the herbicide 2,4-dichlorophenoxyacetic acid. Oral administration of the fractions in a dose of 0.1 mg/kg body weight eliminated the suppressive effect of the herbicide on murine cellular and humoral immune reactions, which manifested by the recovery of the studied parameters to those in control animals.

  14. Assessing the Chemical Accuracy of Protein Structures via Peptide Acidity

    PubMed Central

    Anderson, Janet S.; Hernández, Griselda; LeMaster, David M.

    2012-01-01

    Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses. PMID:23182463

  15. Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

    PubMed

    Kinumi, Tomoya; Goto, Mari; Eyama, Sakae; Kato, Megumi; Kasama, Takeshi; Takatsu, Akiko

    2012-07-01

    A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

  16. Investigating the inclusion properties of aromatic amino acids complexing beta-cyclodextrins in model peptides.

    PubMed

    Caso, Jolanda Valentina; Russo, Luigi; Palmieri, Maddalena; Malgieri, Gaetano; Galdiero, Stefania; Falanga, Annarita; Isernia, Carla; Iacovino, Rosa

    2015-10-01

    Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. β-cyclodextrins (β-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the β-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the β-CD.

  17. Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion.

    PubMed

    Fernandez, Roberto M; Vieira, Renata F F; Nakaie, Clóvis R; Lamy, M Teresa; Ito, Amando S

    2005-01-01

    Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.

  18. Aqueous synthesized near-infrared-emitting quantum dots for RGD-based in vivo active tumour targeting

    NASA Astrophysics Data System (ADS)

    Lu, Yimei; Zhong, Yiling; Wang, Jie; Su, Yuanyuan; Peng, Fei; Zhou, Yanfeng; Jiang, Xiangxu; He, Yao

    2013-04-01

    Over the past two decades, fluorescent quantum dots (QDs) have been highly attractive for a myriad of bioapplications due to their unique optical properties. For bioimaging applications, QD-based in vivo specific tumour targeting is vitally important in the biological and biomedical fields. Aqueous synthesized QDs (aqQDs) exhibit excellent aqueous dispersibility without requiring any post-treatment and have small hydrodynamic diameters (generally <5 nm), which are highly useful for bioimaging applications. We herein present the first example of in vivo active tumour targeting using water-dispersed near-infrared-emitting aqQDs modified with Arg-Gly-Asp (RGD) peptides. In vitro and in vivo studies (e.g., tumour cell labelling, histological analysis, and active tumour targeting) demonstrate that the prepared RGD-decorated aqQDs exhibit highly bio-specific properties, enabling sensitive and specific targeting of tumour sites in both cells and living animals. Our results suggest that the new class of RGD-decorated aqQDs are highly promising as fluorescent bioprobes for a wide range of biological applications.

  19. Bone regeneration potential of stem cells derived from periodontal ligament or gingival tissue sources encapsulated in RGD-modified alginate scaffold.

    PubMed

    Moshaverinia, Alireza; Chen, Chider; Xu, Xingtian; Akiyama, Kentaro; Ansari, Sahar; Zadeh, Homayoun H; Shi, Songtao

    2014-02-01

    Mesenchymal stem cells (MSCs) provide an advantageous alternative therapeutic option for bone regeneration in comparison to current treatment modalities. However, delivering MSCs to the defect site while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated bone regeneration. Here, we tested the bone regeneration capacity of periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) encapsulated in a novel RGD- (arginine-glycine-aspartic acid tripeptide) coupled alginate microencapsulation system in vitro and in vivo. Five-millimeter-diameter critical-size calvarial defects were created in immunocompromised mice and PDLSCs and GMSCs encapsulated in RGD-modified alginate microspheres were transplanted into the defect sites. New bone formation was assessed using microcomputed tomography and histological analyses 8 weeks after transplantation. Results confirmed that our microencapsulation system significantly enhanced MSC viability and osteogenic differentiation in vitro compared with non-RGD-containing alginate hydrogel microspheres with larger diameters. Results confirmed that PDLSCs were able to repair the calvarial defects by promoting the formation of mineralized tissue, while GMSCs showed significantly lower osteogenic differentiation capability. Further, results revealed that RGD-coupled alginate scaffold facilitated the differentiation of oral MSCs toward an osteoblast lineage in vitro and in vivo, as assessed by expression of osteogenic markers Runx2, ALP, and osteocalcin. In conclusion, these results for the first time demonstrated that MSCs derived from orofacial tissue encapsulated in RGD-modified alginate scaffold show promise for craniofacial bone regeneration. This treatment modality has many potential dental and orthopedic applications.

  20. Remote Enantioselection Transmitted by an Achiral Peptide Nucleic Acid Backbone

    NASA Technical Reports Server (NTRS)

    Kozlov, Igor A.; Orgel, Leslie E.; Nielsen, Peter E.

    2000-01-01

    short homochiral segment of DNA into a PNA helix could have guaranteed that the next short segment of DNA to be incorporated would have the same handedness as the first. Once two segments of the same handedness were present, the probability that a third segment would have the same handedness would increase, and so on. Evolution could then slowly dilute out the PNA part. This scenario would ultimately allow the formation of a chiral oligonucleotide by processes that are largely resistant to enantiomeric crossinhibition. It is important to note that the ligation of homochiral dinucleotides on a nucleic acid template would probably be at least as enantiospecific as the reaction that we have studied. The disadvantage of using chiral monomers as components of a replicating system arises from the difficulty of generating a first long homochiral template from a racemic mixture of monomers, although results of experiments designed to overcome this difficulty by employing homochiral tetramers have been reported.l l The probability of obtaining a homochiral n-mer from achiral substrates is approximately 1P-I if the nontemplate-directed extension of the primer is not enantioselective. Hence, it would be very hard to get started with a homochiral 40-mer, for example. No such difficulty exists in a scenario that originates with an achiral genetic material and in which the incorporation of very few chiral monomers in this achiral background gradually progresses towards homochirality. It seems possible that some PNA sequences could act as catalysts, analogous to ribozymes, even though PNA lacks clear metal binding sites. Although such catalysts could not be enantioselective, the incorporation of as few as two chiral nucleotides could then impose chiral specificity on the system. Furthermore, such patch chimeras could help to bridge the gap in catalytic potential between PNA and RNA, while guaranteeing enantioselectivity.

  1. Application of hydrophilic interaction chromatography retention coefficients for predicting peptide elution with TFA and methanesulfonic acid ion-pairing reagents.

    PubMed

    Wujcik, Chad E; Tweed, Joseph; Kadar, Eugene P

    2010-03-01

    Hydrophilic retention coefficients for 17 peptides were calculated based on retention coefficients previously published for TSKgel silica-60 and were compared with the experimental elution profile on a Waters Atlantis HILIC silica column using TFA and methanesulfonic acid (MSA) as ion-pairing reagents. Relative peptide retention could be accurately determined with both counter-ions. Peptide retention and chromatographic behavior were influenced by the percent acid modifier used with increases in both retention and peak symmetry observed at increasing modifier concentrations. The enhancement of net peptide polarity through MSA pairing shifted retention out by nearly five-fold for the earliest eluting peptide, compared with TFA. Despite improvements in retention and efficiency (N(eff)) for MSA over TFA, a consistent reduction in calculated selectivity (alpha) was observed. This result is believed to be attributed to the stronger polar contribution of MSA masking and diminishing the underlying influence of the amino acid residues of each associated peptide. Finally, post-column infusion of propionic acid and acetic acid was evaluated for their potential to recover signal intensity for TFA and MSA counter-ions for LC-ESI-MS applications. Acetic acid generally yielded more substantial signal improvements over propionic acid on the TFA system while minimal benefits and some further reductions were noted with MSA.

  2. Formation pathways and opioid activity data for 3-hydroxypyridinium compounds derived from glucuronic acid and opioid peptides by Maillard processes.

    PubMed

    Horvat, Stefica; Roscić, Maja; Lemieux, Carole; Nguyen, Thi M-D; Schiller, Peter W

    2007-07-01

    The kinetics of formation and identity of the reaction products of the glucuronic acid with three representative opioid peptides were investigated in vitro. Peptides were conjugated with glucuronic acid either in solution or under dry-heating conditions. From the incubations performed in solution N-(1-deoxy-D-fructofuranos-1-yluronic acid)-peptide derivatives (Amadori compounds) were isolated, whereas from the dry-heated reactions products containing the 3-hydroxypyridinium moiety at the N-terminal of the peptide chain were obtained. Experiments performed under mild dry-heating conditions (40 degrees C) in model systems based on Leu-enkephalin and glucuronic acid, and in environment of either 40% or 75% relative humidity, revealed that the higher level of humidity promoted a process that enhanced 3-hydroxypyridinium compound generation. The mechanism of 3-hydroxypyridinium formation is discussed. In comparison with their respective parent peptides, the N-(1-deoxy-D-fructofuranosyl-uronic acid) derivatives of the opioid peptides showed three- to 11-fold lower mu- and delta-receptor-binding affinities and agonist potencies in the functional assays, likely as a consequence of the steric bulk introduced at the N-terminal amino group. The further decrease in opioid activity observed with the 3-hydroxypyridinium-containing peptides may be due to the lower pK(a) of the 3-hydroxypyridinium moiety and to delocalization of the positive charge in the pyridinium ring system.

  3. Engineered peptide-based nanobiomaterials for electrochemical cell chip

    NASA Astrophysics Data System (ADS)

    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.

  4. Anticoagulant Effects of Heparin Complexes with Prolyl-Glycine Peptide and Glycine and Proline Amino Acids.

    PubMed

    Grigorieva, M E; Obergan, T Yu; Maystrenko, E S; Kalugina, M D

    2016-05-01

    The study demonstrates the formation of heparin complexes with prolyl-glycine peptide and proline and glycine amino acids. The method was developed for in vitro production of these complexes at 1:1 dipeptide to heparin molar ratio and 2:1 amino acid to heparin molar ratio. These complexes, unlike the constituents, proline and glycine, exhibited significant anticoagulant, antiplatelet, and fibrin-depolymerization activities of varying degree in vitro and in vivo. The heparin-dipeptide complex produced maximum effect. The dipeptide by itself also showed anticoagulant properties, but less pronounced than in the complex with heparin.

  5. Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake.

    PubMed

    Basu, S; Wickstrom, E

    1997-01-01

    DNA therapeutics show great potential for gene-specific, nontoxic therapy of a wide variety of diseases. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to improve nuclease stability and cell membrane permeability. Recently, a new DNA derivative with an amide backbone instead of a deoxyribose phosphate backbone, peptide nucleic acid (PNA), has shown tremendous potential as an antisense agent. Although PNAs hybridize very strongly and specifically to RNA and DNA, they are taken up by cells very poorly, limiting their potential as nucleic acid binding agents. To improve cellular uptake of a PNA sequence, it was conjugated to a D-amino acid analog of insulin-like growth factor 1 (IGF1), which binds selectively to the cell surface receptor for insulin-like growth factor 1 (IGF1R). The IGF1 D-peptide analog was assembled on (4-methylbenzhydryl)amine resin, and then the PNA was extended as a continuation of the peptide. The conjugate and control sequences were radiolabeled with 14C or fluorescently labeled with fluorescein isothiocyanate. Cellular uptake of the PNA-peptide conjugate, a control with two alanines in the peptide, and a control PNA without the peptide segment were studied in murine BALB/c 3T3 cells, which express low levels of murine IGF1R, in p6 cells, which are BALB/c 3T3 cells which overexpress a transfected human IGF1R gene, and in human Jurkat cells, which do not express IGF1R, as a negative control. The specific PNA-peptide conjugate displayed much higher uptake than the control PNA, but only in cells expressing IGF1R. This approach may allow cell-specific and tissue-specific application of PNAs as gene-regulating agents in vivo.

  6. Amino acid sequence of homologous rat atrial peptides: natriuretic activity of native and synthetic forms.

    PubMed Central

    Seidah, N G; Lazure, C; Chrétien, M; Thibault, G; Garcia, R; Cantin, M; Genest, J; Nutt, R F; Brady, S F; Lyle, T A

    1984-01-01

    A substance called atrial natriuretic factor (ANF), localized in secretory granules of atrial cardiocytes, was isolated as four homologous natriuretic peptides from homogenates of rat atria. The complete sequence of the longest form showed that it is composed of 33 amino acids. The three other shorter forms (2-33, 3-33, and 8-33) represent amino-terminally truncated versions of the 33 amino acid parent molecule as shown by analysis of sequence, amino acid composition, or both. The proposed primary structure agrees entirely with the amino acid composition and reveals no significant sequence homology with any known protein or segment of protein. The short form ANF-(8-33) was synthesized by a multi-fragment condensation approach and the synthetic product was shown to exhibit specific activity comparable to that of the natural ANF-(3-33). PMID:6232612

  7. Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Amino-Thiol Unnatural Amino Acids

    PubMed Central

    Frost, John R.; Jacob, Nicholas T.; Papa, Louis J.; Owens, Andrew E.

    2015-01-01

    A versatile method for orchestrating the formation of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side-chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles. PMID:25933125

  8. Gas-phase Structure and Fragmentation Pathways of Singly Protonated Peptides with N-terminal Arginine

    PubMed Central

    Bythell, Benjamin J.; Csonka, István P.; Suhai, Sándor; Barofsky, Douglas F.; Paizs, Béla

    2010-01-01

    The gas-phase structures and fragmentation pathways of the singly protonated peptide arginylglycylaspartic acid (RGD) are investigated by means of collision-induced-dissociation (CID) and detailed molecular mechanics and density functional theory (DFT) calculations. It is demonstrated that despite the ionizing proton being strongly sequestered at the guanidine group, protonated RGD can easily be fragmented on charge directed fragmentation pathways. This is due to facile mobilization of the C-terminal or aspartic acid COOH protons thereby generating salt-bridge (SB) stabilized structures. These SB intermediates can directly fragment to generate b2 ions or facilely rearrange to form anhydrides from which both b2 and b2+H2O fragments can be formed. The salt-bridge stabilized and anhydride transition structures (TSs) necessary to form b2 and b2+H2O are much lower in energy than their traditional charge solvated counterparts. These mechanisms provide compelling evidence of the role of SB and anhydride structures in protonated peptide fragmentation which complements and supports our recent findings for tryptic systems (Bythell, B. J.; Suhai, S.; Somogyi, A.; Paizs, B. J. Am. Chem. Soc., 2009, 131, 14057–14065.). In addition to these findings we also report on the mechanisms for the formation of the b1 ion, neutral loss (H2O, NH3, guanidine) fragment ions and the d3 ion. PMID:20973555

  9. Acetylation dictates the morphology of nanophase biosilica precipitated by a 14-amino acid leucine-lysine peptide.

    PubMed

    Lutz, Helmut; Jaeger, Vance; Bonn, Mischa; Pfaendtner, Jim; Weidner, Tobias

    2017-02-01

    N-terminal acetylation is a commonly used modification technique for synthetic peptides, mostly applied for reasons of enhanced stability, and in many cases regarded as inconsequential. In engineered biosilification - the controlled deposition of silica for nanotechnology applications by designed peptides - charged groups often play a deciding role. Here we report that changing the charge by acetylation of a 14-amino acid leucine-lysine (LK) peptide dramatically changes the morphology of precipitated biosilica; acetylated LK peptides produce nano-spheres, whereas nano-wires are precipitated by the same peptide in a non-acetylated form. By using interface-specific vibrational spectroscopy and coarse-grained molecular simulations, we show that this change in morphology is not the result of modified peptide-silica interactions, but rather caused by the stabilization of the hydrophobic core of peptide aggregates created by the removal of a peptide charge upon acetylation. These results should raise awareness of the potential impact of N-terminal modifications in peptide applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  10. The Perseus Exobiology mission on MIR: behaviour of amino acids and peptides in Earth orbit.

    PubMed

    Boillot, F; Chabin, A; Buré, C; Venet, M; Belsky, A; Bertrand-Urbaniak, M; Delmas, A; Brack, A; Barbier, B

    2002-08-01

    Leucine, alpha-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and alpha-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection. Analysis of the material after the flight did not reveal any racemization or polymerisation but did provide information regarding photochemical pathways for the degradation of leucine and of the tripeptide. Amino acids appeared to be more sensitive to UV radiation than peptides, the cyclic dipeptide being found to be as particularly resistant. Meteorite powder which exhibits the highest absorption in Vacuum UltraViolet (VUV) afforded the best protection to the organic molecules whereas montmorillonite clay, almost transparent in VUV, was the least efficient. By varying the thickness of the meteorite, we found that the threshold for efficient protection against radiation was about 5 microm. The possible exogenous origin of biological building blocks is discussed with respect to the stability to the molecules and the nature of the associated minerals.

  11. Antitumor magnetic hyperthermia induced by RGD-functionalized Fe3O4 nanoparticles, in an experimental model of colorectal liver metastases

    PubMed Central

    Arriortua, Oihane K; Garaio, Eneko; Herrero de la Parte, Borja; Lezama, Luis; Plazaola, Fernando; García, Jose Angel; Aizpurua, Jesús M; Sagartzazu, Maialen; Irazola, Mireia; Etxebarria, Nestor; García-Alonso, Ignacio; Saiz-López, Alberto; Echevarria-Uraga, José Javier

    2016-01-01

    This work reports important advances in the study of magnetic nanoparticles (MNPs) related to their application in different research fields such as magnetic hyperthermia. Nanotherapy based on targeted nanoparticles could become an attractive alternative to conventional oncologic treatments as it allows a local heating in tumoral surroundings without damage to healthy tissue. RGD-peptide-conjugated MNPs have been designed to specifically target αVβ3 receptor-expressing cancer cells, being bound the RGD peptides by “click chemistry” due to its selectivity and applicability. The thermal decomposition of iron metallo-organic precursors yield homogeneous Fe3O4 nanoparticles that have been properly functionalized with RGD peptides, and the preparation of magnetic fluids has been achieved. The nanoparticles were characterized by transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), electron magnetic resonance (EMR) spectroscopy and magnetic hyperthermia. The nanoparticles present superparamagnetic behavior with very high magnetization values, which yield hyperthermia values above 500 W/g for magnetic fluids. These fluids have been administrated to rats, but instead of injecting MNP fluid directly into liver tumors, intravascular administration of MNPs in animals with induced colorectal tumors has been performed. Afterwards the animals were exposed to an alternating magnetic field in order to achieve hyperthermia. The evolution of an in vivo model has been described, resulting in a significant reduction in tumor viability. PMID:28144504

  12. Programmable Multivalent Display of Receptor Ligands using Peptide Nucleic Acid Nanoscaffolds

    PubMed Central

    Englund, Ethan A.; Wang, Deyun; Fujigaki, Hidetsugu; Sakai, Hiroyasu; Micklitsch, Christopher M.; Ghirlando, Rodolfo; Martin-Manso, Gema; Pendrak, Michael L.; Roberts, David D.; Durell, Stewart R.; Appella, Daniel H.

    2012-01-01

    Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models. PMID:22233624

  13. A descriptor of amino acids: SVRG and its application to peptide quantitative structure-activity relationship.

    PubMed

    Tong, J; Che, T; Li, Y; Wang, P; Xu, X; Chen, Y

    2011-01-01

    In this work, a descriptor, SVRG (principal component scores vector of radial distribution function descriptors and geometrical descriptors), was derived from principal component analysis (PCA) of a matrix of two structural variables of coded amino acids, including radial distribution function index (RDF) and geometrical index. SVRG scales were then applied in three panels of peptide quantitative structure-activity relationships (QSARs) which were modelled by partial least squares regression (PLS). The obtained models with the correlation coefficient (R²(cum)), cross-validation correlation coefficient (Q²(LOO)) were 0.910 and 0.863 for 48 bitter-tasting dipeptides; 0.968 and 0.931 for 21 oxytocin analogues; and 0.992 and 0.954 for 20 thromboplastin inhibitors. Satisfactory results showed that SVRG contained much chemical information relating to bioactivities. The approach may be a useful structural expression methodology for studies on peptide QSAR.

  14. Design, synthesis and biological evaluation of novel non-peptide boronic acid derivatives as proteasome inhibitors.

    PubMed

    Ge, Ying; Li, Aibo; Wu, Jianwei; Feng, Haiwei; Wang, Letian; Liu, Hongwu; Xu, Yungen; Xu, Qingxiang; Zhao, Li; Li, Yuyan

    2017-03-10

    A novel series of non-peptide proteasome inhibitors bearing the 1, 4-naphthoquinone scaffold and boronic acid warhead was developed. In the biological evaluation on the chymotrypsin-like activity of human 20S proteasome, five compounds showed IC50 values in the nanomolar range. Docking experiments into the yeast 20S proteasome rationalized their biological activities and allowed further optimization of this interesting class of inhibitors. Within the cellular proliferation inhibition assay and western blot analysis, compound 3e demonstrated excellent anti-proliferative activity against solid tumor cells and clear accumulation of ubiquitinated cellular proteins. Furthermore, in the microsomal stability assay compound 3e demonstrated much improved metabolic stability compared to bortezomib, emerging as a promising lead compound for further design of non-peptide proteasome inhibitors.

  15. Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

    PubMed Central

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

    A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

  16. Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1981-01-01

    The synthesis of peptides from individual amino acids or pairs of amino acids and ATP in the presence of catalysis by nucleoproteinoid microparticles is investigated. Experiments were performed with suspensions formed from the condensation of lysine-rich and acidic proteinoids with polyadenylic acid, to which were added glycine, phenylalanine, proline, lysine or glycine-phenylalanine mixtures, and ATP either at once or serially. Peptide yields are found to be greatest for equal amounts of acidic and basic proteinoids. The addition of imidazole is found to alter the preference of glycine-phenylalanine mixtures to form mixed heteropeptides rather than homopeptides. A rapid ATP decay in the peptide synthesis reaction is observed, and a greater yield is obtained for repeated small additions than for a single addition of ATP. The experimental system has properties similar to modern cells, and represents an organizational unit ready for the evolution of associated biochemical pathways.

  17. Synthesis of stable C-linked ferrocenyl amino acids and their use in solution-phase peptide synthesis.

    PubMed

    Philip, Anijamol T; Chacko, Shibin; Ramapanicker, Ramesh

    2015-12-01

    Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post-synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C-linked side chain are potentially useful building units for the synthesis of ferrocene-containing peptides. We report here an efficient route to synthesize ferrocene-containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2-ferrocenyl-1,3-dithiane and iodides derived from aspartic acid or glutamic acid using n-butyllithium leads to the incorporation of a ferrocenyl unit to the δ-position or ε-position of an α-amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C-terminus and N-terminus of tripeptides in solution phase.

  18. Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora.

    PubMed

    Didelot, Sandrine; Bordenave-Juchereau, Stephanie; Rosenfeld, Eric; Piot, Jean-Marie; Sannier, Frederic

    2006-05-01

    Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.

  19. Solvation thermodynamics of amino acid side chains on a short peptide backbone

    SciTech Connect

    Hajari, Timir; Vegt, Nico F. A. van der

    2015-04-14

    The hydration process of side chain analogue molecules differs from that of the actual amino acid side chains in peptides and proteins owing to the effects of the peptide backbone on the aqueous solvent environment. A recent molecular simulation study has provided evidence that all nonpolar side chains, attached to a short peptide backbone, are considerably less hydrophobic than the free side chain analogue molecules. In contrast to this, the hydrophilicity of the polar side chains is hardly affected by the backbone. To analyze the origin of these observations, we here present a molecular simulation study on temperature dependent solvation free energies of nonpolar and polar side chains attached to a short peptide backbone. The estimated solvation entropies and enthalpies of the various amino acid side chains are compared with existing side chain analogue data. The solvation entropies and enthalpies of the polar side chains are negative, but in absolute magnitude smaller compared with the corresponding analogue data. The observed differences are large; however, owing to a nearly perfect enthalpy-entropy compensation, the solvation free energies of polar side chains remain largely unaffected by the peptide backbone. We find that a similar compensation does not apply to the nonpolar side chains; while the backbone greatly reduces the unfavorable solvation entropies, the solvation enthalpies are either more favorable or only marginally affected. This results in a very small unfavorable free energy cost, or even free energy gain, of solvating the nonpolar side chains in strong contrast to solvation of small hydrophobic or nonpolar molecules in bulk water. The solvation free energies of nonpolar side chains have been furthermore decomposed into a repulsive cavity formation contribution and an attractive dispersion free energy contribution. We find that cavity formation next to the peptide backbone is entropically favored over formation of similar sized nonpolar side

  20. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  1. Quantitative Analysis of Single Amino Acid Variant Peptides Associated with Pancreatic Cancer in Serum by an Isobaric Labeling Quantitative Method

    PubMed Central

    2015-01-01

    Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research. PMID:25393578

  2. Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1983-01-01

    The kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine, from N-acetylglycyl adenylate anhydride and glycine in aqueous solution at 25 C, and at various PH's are reported. The reaction is of interest in that over a physiologically relevant pH range (6-8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrlysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

  3. Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

    NASA Astrophysics Data System (ADS)

    Abutokaikah, Maha T.; Guan, Shanshan; Bythell, Benjamin J.

    2017-01-01

    Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra.

  4. In Vitro Assessment of a Peptide Nucleic Acid (PNA) - Peptide Conjugate Labeled With an Auger-Emitting Radionuclide for Prostate Cell Killing

    DTIC Science & Technology

    2005-02-01

    synthesis of a peptide nucleic acid (PNA) that has an Auger-emitter (1-125) incorporated. By design the PNA will bind with mRNA and DNA associated with...bind with cell surface gastrin -releasing peptide receptors and be internalized (3). Binding with mRNA and nuclear DNA specific to the insulin-like...route proposed to prepare 10 is shown in Figure 1 (compounds 1-10). This synthesis began with the preparation of the base-reactive intermediate 5

  5. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide

    PubMed Central

    Abushahba, Mostafa FN; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  6. Importance of backbone angles versus amino acid configurations in peptide vibrational Raman optical activity spectra

    NASA Astrophysics Data System (ADS)

    Herrmann, Carmen; Ruud, Kenneth; Reiher, Markus

    2008-01-01

    In this work, we investigate whether the differential scattering of right- and left-circularly polarized light in peptide Raman optical activity spectra are uniquely dominated by the backbone conformation, or whether the configurations of the individual amino acid also play a significant role. This is achieved by calculating Raman optical activity spectra using density functional theory for four structurally related peptides with a common backbone conformation, but with different sequences of amino acid configurations. Furthermore, the ROA signals of the amide normal modes are decomposed into contributions from groups of individual atoms. It is found that the amino acid configuration has a considerable influence on the ROA peaks in the amide I, II, and III regions, although the local decomposition reveals that the side-chain atoms only contribute to those peaks directly in the case of the amide II vibrations. Furthermore, small changes in the amide normal modes may lead to large and irregular modifications in the ROA intensity differences, making it difficult to establish transferable ROA intensity differences even for structurally similar vibrations.

  7. Cα-C bond cleavage of the peptide backbone in MALDI in-source decay using salicylic acid derivative matrices.

    PubMed

    Asakawa, Daiki; Takayama, Mitsuo

    2011-07-01

    The use of 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA) as novel matrices for in-source decay (ISD) of peptides in matrix-assisted laser desorption/ionization (MALDI) is described. The use of 5-FSA and 5-NSA generated a- and x-series ions accompanied by oxidized peptides [M - 2 H + H](+). The preferential formation of a- and x-series ions was found to be dependent on the hydrogen-accepting ability of matrix. The hydrogen-accepting ability estimated from the ratio of signal intensity of oxidized product [M - 2 H + H](+) to that of non-oxidized protonated molecule [M + H](+) of peptide was of the order 5-NSA > 5-FSA > 5-aminosalicylic acid (5-ASA) ≒ 2,5-dihydroxyl benzoic acid (2,5-DHB) ≒ 0. The results suggest that the hydrogen transfer reaction from peptide to 5-FSA and 5-NSA occurs during the MALDI-ISD processes. The hydrogen abstraction from peptides results in the formation of oxidized peptides containing a radical site on the amide nitrogen with subsequent radical-induced cleavage at the Cα-C bond, leading to the formation of a- and x-series ions. The most significant feature of MALDI-ISD with 5-FSA and 5-NSA is the specific cleavage of the Cα-C bond of the peptide backbone without degradation of side-chain and post-translational modifications (PTM). The matrix provides a useful complementary method to conventional MALDI-ISD for amino acid sequencing and site localization of PTMs in peptides.

  8. Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

    PubMed

    Sholokh, Marianna; Zamotaiev, Oleksandr M; Das, Ranjan; Postupalenko, Viktoriia Y; Richert, Ludovic; Dujardin, Denis; Zaporozhets, Olga A; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2015-02-12

    Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

  9. Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids.

    PubMed

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2014-01-01

    Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

  10. Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

    PubMed Central

    Feagin, Trevor A.; Shah, Nirmal I.; Heemstra, Jennifer M.

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  11. Room temperature N-arylation of amino acids and peptides using copper(I) and β-diketone.

    PubMed

    Sharma, Krishna K; Sharma, Swagat; Kudwal, Anurag; Jain, Rahul

    2015-04-28

    A mild and efficient method for the N-arylation of zwitterionic amino acids, amino acid esters and peptides is described. The procedure provides the first room temperature synthesis of N-arylated amino acids and peptides using CuI as a catalyst, diketone as a ligand, and aryl iodides as coupling partners. The method is equally applicable for using relatively inexpensive aryl bromides as coupling partners at 80 °C. Using this procedure, electronically and sterically diverse aryl halides, containing reactive functional groups were efficiently coupled in good to excellent yields.

  12. Topical Delivery of Hyaluronic Acid into Skin using SPACE-peptide Carriers

    PubMed Central

    Chen, Ming; Gupta, Vivek; Anselmo, Aaron C.; Muraski, John A.; Mitragotri, Samir

    2014-01-01

    Topical penetration of macromolecules into skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200–325 kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110 nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/−1.1-fold compared to PBS. The system also enhanced penetration of HA in human skin in vitro, penetrating deep into the epidermis and dermis in skin of both species. In vivo experiments performed using SKH1 hairless mice also confirmed increased dermal penetration of HA using the delivery system; a 5-fold enhancement in penetration was found compared to PBS control. Concentrations of HA in skin were about 1000-fold higher than those in blood; confirming the localized nature of HA delivery into skin. The SPACE-ethosomal delivery system provides a formulation for topical delivery of macromolecules that are otherwise difficult to deliver into skin. PMID:24129342

  13. Black mamba venom peptides target acid-sensing ion channels to abolish pain.

    PubMed

    Diochot, Sylvie; Baron, Anne; Salinas, Miguel; Douguet, Dominique; Scarzello, Sabine; Dabert-Gay, Anne-Sophie; Debayle, Delphine; Friend, Valérie; Alloui, Abdelkrim; Lazdunski, Michel; Lingueglia, Eric

    2012-10-25

    Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

  14. Novel poly(ethylene-co-acrylic acid) nanofibrous biomaterials for peptide synthesis and biomedical applications.

    PubMed

    Xiang, Bei; Sun, Gang; Lam, Kit S; Xiao, Kai

    2010-10-01

    Poly(ethylene-co-acrylic acid) (PE-co-AA) fibers in sizes of 200-500 nm were prepared by using a novel melt-extrusion-extraction fabrication process. The thermoplastic nanofibers could be controllably dispersed and reassembled by a novel solvent exchange filtration method. The dispersed PE-co-AA nanofibers possess active surface areas and could directly conduct chemical reactions on surfaces. Surface modifications and organic synthesis on the nanofibers were proven effective and controllable after the dispersion. Multistep synthesis of biomolecules, such as peptide ligand HWRGWV against Fc portion of human IgG, was successful. The surface-anchored ligand has shown bioactivity through selective binding to and staining by human IgG-alkaline phosphatase conjugate. Another peptide, LXY3, a selective cyclic peptide ligand against alpha3beta1 integrin of MDA-MB-231 breast cancer cells, was also prepared on the surfaces of the dispersed nanofibers. The results showed that MDA-MB-231 cells were able to specifically bind to and grow on surfaces of the nanofibers that were functionalized with LXY3.

  15. Differentiating amino acid residues and side chain orientations in peptides using scanning tunneling microscopy.

    PubMed

    Claridge, Shelley A; Thomas, John C; Silverman, Miles A; Schwartz, Jeffrey J; Yang, Yanlian; Wang, Chen; Weiss, Paul S

    2013-12-11

    Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structures at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer's and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level.

  16. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.

    PubMed

    Bieling, Peter; Beringer, Malte; Adio, Sarah; Rodnina, Marina V

    2006-05-01

    Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).

  17. Influence of the yeast strain on the changes of the amino acids, peptides and proteins during sparkling wine production by the traditional method.

    PubMed

    Martínez-Rodríguez, A J; Carrascosa, A V; Martín-Alvarez, P J; Moreno-Arribas, V; Polo, M C

    2002-12-01

    The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions.

  18. Fermentation of peptides and amino acids by a monensin-sensitive ruminal Peptostreptococcus.

    PubMed Central

    Chen, G J; Russell, J B

    1988-01-01

    A monensin-sensitive ruminal peptostreptococcus was able to grow rapidly (growth rate of 0.5/h) on an enzymatic hydrolysate of casein, but less than 23% of the amino acid nitrogen was ever utilized. When an acid hydrolysate was substituted for the enzymatic digest, more than 31% of the nitrogen was converted to ammonia and cell protein. Coculture experiments and synergisms with peptide-degrading strains of Bacteroides ruminicola and Streptococcus bovis indicated that the peptostreptococcus was unable to transport certain peptides or hydrolyze them extracellularly. Leucine, serine, phenylalanine, threonine, and glutamine were deaminated at rates of 349, 258, 102, 95, and 91 nmol/mg of protein per min, respectively. Deamination rates for some other amino acids were increased when the amino acids were provided as pairs of oxidized and reduced amino acids (Stickland reactions), but these rates were still less than 80 nmol/mg of protein per min. In continuous culture (dilution rate of 0.1/h), bacterial dry matter and ammonia production decreased dramatically at a pH of less than 6.0. When dilution rates were increased from 0.08 to 0.32/h (pH 7.0), ammonia production increased while production of bacterial dry matter and protein decreased. These rather peculiar kinetics resulted in a slightly negative estimate of maintenance energy and could not be explained by a change in fermentation products. Approximately 80% of the cell dry matter was protein. When corrections were made for cell composition, the yield of ATP was higher than the theoretical maximum value. It is possible that mechanisms other than substrate-level phosphorylation contributed to the energetics of growth. PMID:2975156

  19. Radical S-adenosyl methionine epimerases: regioselective introduction of diverse D-amino acid patterns into peptide natural products.

    PubMed

    Morinaka, Brandon I; Vagstad, Anna L; Helf, Maximilian J; Gugger, Muriel; Kegler, Carsten; Freeman, Michael F; Bode, Helge B; Piel, Jörn

    2014-08-04

    PoyD is a radical S-adenosyl methionine epimerase that introduces multiple D-configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post-translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D-amino acid patterns into all-L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.

  20. PEI-g-PEG-RGD/Small Interference RNA Polyplex-Mediated Silencing of Vascular Endothelial Growth Factor Receptor and Its Potential as an Anti-Angiogenic Tumor Therapeutic Strategy

    PubMed Central

    Kim, Jihoon; Kim, Sung Wan

    2011-01-01

    Tumor angiogenesis appears to be achieved by the expression of vascular endothelial growth factor (VEGF) within solid tumors that stimulate host vascular endothelial cell mitogenesis and possibly chemotaxis. VEGF's angiogenic actions are mediated through its high-affinity binding to 2 endothelium-specific receptor tyrosine kinase, Flt-1 (VEGFR1), and Flk-1/KDR (VEGFR2). RNA interference-mediated knockdown of protein expression at the messenger RNA level provides a new therapeutic strategy to overcome various diseases. To achieve high efficacy in RNA interference-mediated therapy, it is critical to develop an efficient delivering system to deliver small interference RNA (siRNA) into tissues or cells site-specifically. We previously reported an angiogenic endothelial cell-targeted polymeric gene carrier, PEI-g-PEG-RGD. This targeted carrier was developed by the conjugation of the ανβ3/ανβ5 integrin-binding RGD peptide (ACDCRGDCFC) to the cationic polymer, branched polyethylenimine, with a hydrophilic polyethylene glycol (PEG) spacer. In this study, we used PEI-g-PEG-RGD to deliver siRNA against VEGFR1 into tumor site. The physicochemical properties of PEI-g-PEG-RGD/siRNA complexes was evaluated. Further, tumor growth profile was also investigated after systemic administration of PEI-g-PEG-RGD/siRNA complexes. PMID:21375397

  1. Radiolabeled cyclic arginine-glycine-aspartic (RGD)-conjugated iron oxide nanoparticles as single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) dual-modality agents for imaging of breast cancer

    NASA Astrophysics Data System (ADS)

    Deng, Shengming; Zhang, Wei; Zhang, Bin; Hong, Ruoyu; Chen, Qing; Dong, Jiajia; Chen, Yinyiin; Chen, Zhiqiang; Wu, Yiwei

    2015-01-01

    Ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) modified with a novel cyclic arginine-glycine-aspartate (RGD) peptide were made and radiolabeled as single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) dual-modality agents for imaging of breast cancer. The probe was tested both in vitro and in vivo to determine its receptor targeting efficacy and feasibility for SPECT and MRI. The radiochemical syntheses of 125I-cRGD-USPIO were accomplished with a radiochemical purity of 96.05 ± 0.33 %. High radiochemical stability was found in fresh human serum and in phosphate-buffered saline. The average hydrodynamic size of 125I-cRGD-USPIO determined by dynamic light scattering was 51.3 nm. Results of in vitro experiments verified the specificity of the radiolabeled nanoparticles to tumor cells. Preliminary biodistribution studies of 125I-radiolabeled cRGD-USPIO in Bcap37-bearing nude mice showed that it had long circulation half-life, high tumor uptake, and high initial blood retention with moderate liver uptake. In vivo tumor targeting and uptake of the radiolabeled nanoparticles in mice model were visualized by SPECT and MRI collected at different time points. Our results strongly indicated that the 125I-cRGD-USPIO could be used as a promising bifunctional radiotracer for early clinical tumor detection with high sensitivity and high spatial resolution by SPECT and MRI.

  2. Purification and amino acid composition of a peptide with molt-inhibiting activity from the lobster, Homarus americanus.

    PubMed

    Chang, E S; Bruce, M J; Newcomb, R W

    1987-01-01

    A peptide was isolated and purified from sinus glands of the lobster, Homarus americanus, that was able to decrease circulating titers of ecdysteroids and increase the molt interval of eyestalk-ablated juvenile lobsters. This molt-inhibiting activity was demonstrated to consist of two very closely related peptides by means of high-performance liquid chromatography and gel electrophoresis. By means of amino acid analyses, a molecular weight of approximately 8700 was obtained.

  3. Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles

    PubMed Central

    2011-01-01

    Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids. PMID:21867548

  4. Beta2-amino acids-syntheses, occurrence in natural products, and components of beta-peptides1,2.

    PubMed

    Lelais, Gérald; Seebach, Dieter

    2004-01-01

    Although they are less abundant than their alpha-analogues, beta-amino acids occur in nature both in free form and bound to peptides. Oligomers composed exclusively of beta-amino acids (so-called beta-peptides) might be the most thoroughly investigated peptidomimetics. Beside the facts that they are stable to metabolism, exhibit slow microbial degradation, and are inherently stable to proteases and peptidases, they fold into well-ordered secondary structures consisting of helices, turns, and sheets. In this respect, the most intriguing effects have been observed when beta2-amino acids are present in the beta-peptide backbone. This review gives an overview of the occurrence and importance of beta2-amino acids in nature, placing emphasis on the metabolic pathways of beta-aminoisobutyric acid (beta-Aib) and the appearance of beta2-amino acids as secondary metabolites or as components of more complex natural products, such as peptides, depsipeptides, lactones, and alkaloids. In addition, a compilation of the syntheses of both achiral and chiral beta2-amino acids is presented. While there are numerous routes to achiral beta2-amino acids, their EPC synthesis is currently the subject of many investigations. These include the diastereoselective alkylation and Mannich-type reactions of cyclic- or acyclic beta-homoglycine derivatives containing chiral auxiliaries, the Curtius degradation, the employment of transition-metal catalyzed reactions such as enantioselective hydrogenations, reductions, C-H insertions, and Michael-type additions, and the resolution of rac. beta2-amino acids, as well as several miscellaneous methods. In the last part of the review, the importance of beta2-amino acids in the formation of beta-peptide secondary structures is discussed.

  5. Information transfer from peptide nucleic acids to RNA by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.

  6. Preparation of surfactant-stabilized gold nanoparticle-peptide nucleic acid conjugates

    NASA Astrophysics Data System (ADS)

    Duy, Janice; Connell, Laurie B.; Eck, Wolfgang; Collins, Scott D.; Smith, Rosemary L.

    2010-09-01

    A simple, two-step method of producing stable and functional peptide nucleic acid (PNA)-conjugated gold nanoparticles using a surfactant stabilization step is presented. PNA are DNA analogs with superior chemical stability and target discrimination, but their use in metallic nanoparticle systems has been limited by the difficulty of producing stable colloids of nanoparticle-PNA conjugates. In this work, the nonionic surfactant Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was used to sterically shield gold surfaces prior to the addition of thiolated PNA, producing conjugates which remain dispersed in solution and retain the ability to hybridize to complementary nucleic acid sequences. The conjugates were characterized using transmission electron microscopy, dynamic light scattering, and UV-visible absorbance spectroscopy. PNA attachment to gold nanoparticles was confirmed with an enzyme-linked immunoassay, while the ability of nanoparticle-bound PNA to hybridize to its complement was demonstrated using labeled DNA.

  7. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

    PubMed Central

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  8. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines.

    PubMed

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  9. An extract of Gymnema sylvestre leaves and purified gymnemic acid inhibits glucose-stimulated gastric inhibitory peptide secretion in rats.

    PubMed

    Fushiki, T; Kojima, A; Imoto, T; Inoue, K; Sugimoto, E

    1992-12-01

    Gastric inhibitory peptide release into the portal vein in response to duodenal infusion of D-glucose was studied in the presence of a leaf extract of Gymnema sylvestre, purified gymnemic acid and inhibitors of some putative glucose sensors and carriers in the intestinal lumen. Intraduodenal infusion of D-glucose significantly increased the portal immunoreactive gastric inhibitory peptide concentration in a dose-dependent manner. The increase in the portal immunoreactive gastric inhibitory peptide induced by glucose was significantly depressed by concomitantly infused leaf extract of Gymnema sylvestre, purified gymnemic acid and phlorizin but not by cytochalasin B. Mannoheptulose, which inhibits glycolysis, and procaine and lidocaine, which inhibit the vagal glucoreceptor in the lumen, did not affect portal immunoreactive gastric inhibitory peptide concentrations. These results suggest that a glucose receptor, which interacts with the leaf extract of Gymnema sylvestre, purified gymnemic acid and phlorizin, exists for the release of immunoreactive gastric inhibitory peptide and that the glucose receptor for gastric inhibitory peptide release is not likely to be identical with a glucose transporter or a vagal glucoreceptor in the lumen.

  10. Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

    PubMed

    Simonson, Oscar E; Svahn, Mathias G; Törnquist, Elisabeth; Lundin, Karin E; Smith, C I E

    2005-01-01

    Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

  11. In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.

    PubMed

    Beavers, Kelsey R; Mares, Jeremy W; Swartz, Caleb M; Zhao, Yiliang; Weiss, Sharon M; Duvall, Craig L

    2014-07-16

    Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA-PSi platform with broad utility in drug delivery and biosensing.

  12. Peptide nucleic acids rather than RNA may have been the first genetic molecule

    NASA Technical Reports Server (NTRS)

    Nelson, K. E.; Levy, M.; Miller, S. L.

    2000-01-01

    Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

  13. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    PubMed Central

    Braga, Daniel; Hoffmeister, Dirk

    2016-01-01

    Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis. PMID:28144348

  14. Conformational characterization of the 1-aminocyclobutane-1-carboxylic acid residue in model peptides.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Toniolo, C; Bonora, G M; Benedetti, Z; Di Blasio, B; Iacovino, R; Santini, A; Saviano, M; Kamphuis, J

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined.

  15. Identification of laminin α5 short arm peptides active for endothelial cell attachment and tube formation.

    PubMed

    Kikkawa, Yamato; Sugawara, Yumika; Harashima, Nozomi; Fujii, Shogo; Ikari, Kazuki; Kumai, Jun; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi

    2017-02-21

    Laminin-511, a major component of endothelial basement membrane, consists of α5, β1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  16. Role of SbmA in the uptake of peptide nucleic acid (PNA)-peptide conjugates in E. coli.

    PubMed

    Ghosal, Anubrata; Vitali, Ally; Stach, James E M; Nielsen, Peter E

    2013-02-15

    Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-D((KFF)(3)K) peptide retained antibacterial activity in sbmA deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-D((KFF)(3)K), H-(RFR)(4)-Ahx-βAla, H-(R-Ahx-R)(4)-Ahx-βAla, and H-(R-Ahx)(6)-βAla, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA.

  17. An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3- mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation

    PubMed Central

    1991-01-01

    The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well- defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface- bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required. PMID:1714913

  18. Antiangiogenic gene therapy of experimental pancreatic tumor by sFlt-1 plasmid DNA carried by RGD-modified crosslinked polyplex micelles.

    PubMed

    Vachutinsky, Yelena; Oba, Makoto; Miyata, Kanjiro; Hiki, Shigehiro; Kano, Mitsunobu R; Nishiyama, Nobuhiro; Koyama, Hiroyuki; Miyazono, Kohei; Kataoka, Kazunori

    2011-01-05

    Disulfide crosslinked polyplex micelles with RGD peptides were formed through ion complexation of thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(L-lysine) (c(RGDfK)-PEG-P(Lys-SH)) and plasmid DNA encoding sFlt-1 and tested for their therapeutic effect in BxPC3 pancreatic adenocarcinoma tumor bearing mice. These micelles, systemically injected, demonstrated significant inhibition of tumor growth up to day 18, as a result of the antiangiogenic effect that was confirmed by vascular density measurements. Significant therapeutic activity of the 15% crosslinked micelle (c(RGDfK)-PEG-P(Lys-SH15)) was achieved by combined effect of increased tumor accumulation, interaction with endothelial cells and enhanced intracellular uptake through receptor-mediated endocytosis. These results suggest that RGD targeted crosslinked polyplex micelles can be effective plasmid DNA carriers for antiangiogenic gene therapy.

  19. Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using {gamma}-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the {gamma}-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic

  20. Slow peptide bond formation by proline and other N-alkylamino acids in translation

    PubMed Central

    Pavlov, Michael Y.; Watts, Richard E.; Tan, Zhongping; Cornish, Virginia W.; Ehrenberg, Måns; Forster, Anthony C.

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid (“imino acid”), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3–6 times slower than Phe incorporation from Phe-tRNAPhe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNAPro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the “tRNA abundance” hypothesis. PMID:19104062

  1. Hydration studies of electrospray ions from amino acids and small peptides

    NASA Astrophysics Data System (ADS)

    Nguyen, Chuong (Steve)

    This project was undertaken to gain a better understanding of the hydration behaviors of gas phase ions from solutions containing amino acids and peptides. In order to characterize their hydration behavior, the molecules of interest in solutions were first converted into gas phase ions by electrospray ionization (ESI). The completely desolvated ions were then deliberately dispersed into an inert bath gas, usually nitrogen, containing accurately known concentrations of solvent vapor. The resulting mixtures of ions and bath gas were subsequently passed into a vacuum chamber by way of an adiabatic supersonic free jet expansion. The cooling during that expansion caused solvation of the ions, the extent of which was determined by a quadrupole mass analyzer. Mass analysis of the solute ions in the absence of vapor showed peaks with the mass to charge ratios corresponding to the desolvated ions. On the other hand, mass spectrometric analyses of ions in the presence of solvent vapor showed sequences of peaks corresponding to the solvated ions with varying numbers of water molecules. The extent of the ion solvation was controlled by varying the concentration of solvent vapor in the bath gas. Two different scales were proposed for the evaluation of the relative affinities of amino acids for water molecules. One was based primarily on the assumption that the affinities of amino acids for water molecules are directly proportional to their gas phase solvation rate constants ( k). An alternative approach produced an affinity scale based on the extent of ion hydration occurred during the free jet expansion. It was found that the addition of a polar solvent vapor to the bath gas at low concentrations substantially enhanced the production of the bare solute ions from the evaporating charged droplets. This remarkable result not only provided a means to increase the ion production and thus detection sensitivity of mass spectrometric analyses, but also yielded important information

  2. Acidity and metal (Mg2+, Ca2+, Zn2+) affinity of L-γ-carboxyglutamic acid and its peptide analog

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

    2014-10-01

    Density functional theory methods with the B3LYP and B97D functionals with triple-zeta 6-311++G(d,p) basis set have been used to study the acidity, basicity and metal affinity of L-γ-carboxyglutamic acid (GLA) and its peptide derivative [2-acetylamino-3-(methylamino)-3-oxopropyl]malonic acid (AMD-GLA). The Gibbs interaction energies of the GLA2-…M2+ and AMD-GLA2-…M2+ (M = Mg, Ca, Zn) complexes show an increasing binding affinity in the order Ca2+ < Mg2+ < Zn2+ The transition metal Zn2+ is most effectively recognized by the dianions of GLA and AMD-GLA. Of the dianions studied the AMD-GLA dianion is the strongest Lewis base. Computations that include the effect of solvation showed that in water the relative stability of GLA2-…M2+ and AMD-GLA2-…M2+ ionic bonds is rapidly diminished. The computed interaction Gibbs energy in water is small and negative.

  3. Ion-pair mediated transport of small model peptides in liquid phase micro extraction under acidic conditions.

    PubMed

    Reubsaet, J Léon E; Paulsen, Jonas V

    2005-02-01

    This paper discusses the behaviour of five small model peptides in a three phase (aqueous donor-organic-aqueous acceptor) liquid phase micro extraction system in relation to their physico-chemical properties (charge, hydrophobicity). It is proved that for all peptides transport over the organic phase is mediated by aliphatic sulphonic acids. Heptane-1-sulphonic acid gave the best overall recoveries. It appeared that peptides with hydrophobic properties (IPI) and a high number of positive charges (KYK) show good recoveries and are enriched in the acceptor phase. Variation in the pH (1.6-4.4) of the donor phase shows that there are peptide-dependent optimal pH-values for their recovery. Increasing pH in the acceptor phase shows that in most cases the recovery decreases due to decreased ion-pair mediated membrane transport. For KYK the partition between the organic phase and the aqueous acceptor-phase is also driven by the solubility in the aqueous acceptor phase. Increase of the ion strength of the acceptor phase did not affect the recovery of the peptides. Except for KYK, which showed decreased recovery when the ion strength increased. Another finding is that delocalisation of positive charge causes bad recovery, probably due to incomplete ion-pair-peptide complex formation.

  4. Replacement of the Lys linker with an Arg linker resulting in improved melanoma uptake and reduced renal uptake of Tc-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptide.

    PubMed

    Yang, Jianquan; Guo, Haixun; Padilla, R Steve; Berwick, Marianne; Miao, Yubin

    2010-09-15

    The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (alpha-MSH) hybrid peptide through structural modification or L-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), D-Phe7, Arg11] alpha-MSH3-13 {(Arg11)CCMSH} through the Arg linker (substituting the Lys linker) to generate a novel RGD-Arg-(Arg11)CCMSH hybrid peptide. The melanoma targeting and pharmacokinetic properties of 99mTc-RGD-Arg-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The effect of L-lysine co-injection on the renal uptake was determined through the co-injection of L-lysine with 99mTc-RGD-Arg-(Arg11)CCMSH or 99mTc-RGD-Lys-(Arg11)CCMSH. Replacement of the Lys linker with an Arg linker exhibited a profound effect in reducing the non-specific renal uptake of 99mTc-RGD-Arg-(Arg11)CCMSH, as well as increasing the tumor uptake of 99mTc-RGD-Arg-(Arg11)CCMSH compared to 99mTc-RGD-Lys-(Arg11)CCMSH. 99mTc-RGD-Arg-(Arg11)CCMSH exhibited high tumor uptake (21.41+/-3.74% ID/g at 2 h post-injection) and prolonged tumor retention (6.81+/-3.71% ID/g at 24 h post-injection) in B16/F1 melanoma-bearing mice. The renal uptake values of 99mTc-RGD-Arg-(Arg11)CCMSH were 40.14-64.08% of those of 99mTc-RGD-Lys-(Arg11)CCMSH (p<0.05) at 0.5, 2, 4 and 24 h post-injection. Co-injection of L-lysine was effective in decreasing the renal uptakes of 99mTc-RGD-Arg-(Arg11)CCMSH by 27.7% and 99mTc-RGD-Lys-(Arg11)CCMSH by 52.1% at 2 h post-injection. Substitution of the Lys linker with an Arg linker dramatically improved the melanoma uptake and reduced the renal uptake of 99mTc-RGD-Arg-(Arg11)CCMSH, warranting the further evaluation of 188Re-labeled RGD-Arg-(Arg11)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.

  5. Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

    PubMed

    Burster, Timo; Marin-Esteban, Viviana; Boehm, Bernhard O; Dunn, Shannon; Rotzschke, Olaf; Falk, Kirsten; Weber, Ekkehard; Verhelst, Steven H L; Kalbacher, Hubert; Driessen, Christoph

    2007-11-15

    Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

  6. The Prebiotic C-Terminal Elongation of Peptides can be Initiated by N-Carbamoyl Amino Acids.

    PubMed

    Abou Mrad, Ninette; Ajram, Ghinwa; Rossi, Jean-Christophe; Boiteau, Laurent; Duvernay, Fabrice; Pascal, Robert; Danger, Gregoire

    2017-04-05

    The formation of peptides upon EDC promoted activation of N-carbamoylamino acids (CAA), was considered in the scope of our recent works on carbodiimide promoted C-terminus elongation of peptides in a prebiotic context. Thus EDC promoted activation of CAA derivatives of Tyr(Me) or Ala in dilute aqueous medium pH 5.5-6.5 in the presence of excess of AA, resulted in peptide formation via C-terminus activation / elongation. Kinetic results similar to those of EDC-mediated activation of N-acyl-AA lead us to postulate the formation of a 2-amino-5(4H)-oxazolone intermediate by cyclization of the activated CAA, in spite of the absence of epimerization occurred at CAA residues. Thus, in a prebiotic context, CAA may have played a similar role as N-acyl-AA in the initiation of C-terminus peptide elongation.

  7. Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides.

    PubMed

    Vardy, Eyal; Sassano, Maria F; Rennekamp, Andrew J; Kroeze, Wesley K; Mosier, Philip D; Westkaemper, Richard B; Stevens, Craig W; Katritch, Vsevolod; Stevens, Raymond C; Peterson, Randall T; Roth, Bryan L

    2015-06-18

    It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here, we test this idea by comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. The insensitivity of the delta OR (DOR) to deltorphin was due to variation of a single amino acid, Trp7.35, which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish, and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides.

  8. β-Amino acids containing peptides and click-cyclized peptide as β-turn mimics: a comparative study with 'conventional' lactam- and disulfide-bridged hexapeptides.

    PubMed

    Larregola, Maud; Lequin, Olivier; Karoyan, Philippe; Guianvarc'h, Dominique; Lavielle, Solange

    2011-09-01

    The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β-turn stabilization of different analogs designed as mimics of the β-turn structure found in tendamistat. The β-turn conformation of linear β-amino acid-containing peptides and triazole-cyclized analogs were compared to 'conventional' lactam- and disulfide-bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β-turns in solution, and for those not structured in solution in the presence of α-amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac-Ser-Trp-Arg-Tyr-NH(2) and both the amide bond-cyclized, c[Pro-Ser-Trp-Arg-Tyr-D-Ala] and the disulfide-bridged, Ac-c[Cys-Ser-Trp-Arg-Tyr-Cys]-NH(2) hexapeptides adopt dominantly in solution a β-turn conformation closely related to the one observed in tendamistat. On the contrary, the β-amino acid-containing peptides such as Ac-(R)-β(3) -hSer-(S)-Trp-(S)-β(3) -hArg-(S)-β(3) -hTyr-NH(2) , and the triazole cyclic peptide, c[Lys-Ser-Trp-Arg-Tyr-βtA]-NH(2) , both specifically designed to mimic this β-turn, do not adopt stable structures in solution and do not show any characteristics of β-turn conformation. However, these unstructured peptides specifically interact in the active site of α-amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α-amylase inhibitor. Thus, in contrast to amide-cyclized or disulfide-bridged hexapeptides, β-amino acid-containing peptides and click-cyclized peptides may not be regarded as β-turn stabilizers, but can be considered as potential β-turn inducers.

  9. Gamma Peptide Nucleic Acids: As Orthogonal Nucleic Acid Recognition Codes for Organizing Molecular Self-Assembly.

    PubMed

    Sacui, Iulia; Hsieh, Wei-Che; Manna, Arunava; Sahu, Bichismita; Ly, Danith H

    2015-07-08

    Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host's genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

  10. Pipa carvalhoi skin secretion profiling: absence of peptides and identification of kynurenic acid as the major constitutive component.

    PubMed

    Mariano, Douglas Oscar Ceolin; Yamaguchi, Lydia Fumiko; Jared, Carlos; Antoniazzi, Marta Maria; Sciani, Juliana Mozer; Kato, Massuo Jorge; Pimenta, Daniel Carvalho

    2015-01-01

    The presence of peptides has been identified in all African pipid genera; nevertheless, little is known about skin secretion of South American frog genus Pipa. Skin secretion from captive and wild Pipa carvalhoi were obtained in the presence or absence of norepinephrine stimulation. The <10 kDa fraction was analyzed by liquid chromatography and mass spectrometry, searching for peptides. Chromatographic profiles show the presence of a major component in this secretion, regardless of the stimulation method (norepinephrine or mechanical stimulation) and the origin of the animal (captivity or wild), as well as in the absence of any stimulus. The general mass distribution profile in P. carvalhoi skin secretion shows numerous components below 800 Da. Moreover, no peptide could be identified, regardless of the chromatographic approach. The major component was purified and identified as kynurenic acid, an L-tryptophan derivative. P. carvalhoi does not secrete peptides as toxins in its skin. In addition, we here report that kynurenic acid is the main component of P. carvalhoi skin secretion. Although no biological activity was associated with kynurenic acid, we propose that this molecule is a pheromone that signals the presence of a co-specific in the shady environment in which this animal lives. In this study we demonstrate the absence of peptidic toxins in the skin secretion of P. carvalhoi, a break of paradigm in the pipid family.

  11. Rhizobins, a Group of Peptides in the Free-Amino-Acid Pool of the Soybean-Rhizobium System †

    PubMed Central

    Garay, Andrew S.; Ahlgren, Joy A.; Gonzalez, Mark A.; Stasney, Mark A.; Madtes, Paul C.

    1986-01-01

    Free-living Rhizobium (according to Bergey's Manual of Systematic Bacteriology, [1984, The Williams & Wilkins Co., Baltimore], Bradyrhizobium) japonicum was found to release a peptide into the nutrient media. Soybean nodules contained this peptide and exuded it into the soil. The name “rhizobin A” is suggested for this peptide. Nodules also contained another peptide, rhizobin B, as well as an unidentified, ninhydrin-positive compound, rhizobin C. The three peptides were confined to the free-amino-acid pool of the soluble fraction and eluted consecutively from a cation-exchange column. Rhizobin A was isolated in a highly purified form; its molecular mass was approximately 1,600 daltons as determined by Sephadex gel filtration and mass spectrometry. The amino-acid composition could be determined only approximately, because a long time was necessary for acid hydrolysis, possibly due to unusual linkages. The rhizobin concentration in soybean nodules continually increased during 50 days of growth, from 2 to approximately 400 μg/g (fresh weight). When combined nitrogen was added to nodulated soybean and subsequently removed, nitrogenase activity, nodulation, and nodule growth first decreased and then recovered. The relative amount of rhizobin A followed a similar pattern. Rhizobins were not detected in the roots, stems, and leaves of nodulated soybean plants. They were present in Lupinus nodules, but absent in alder nodules. PMID:16347004

  12. Effect of environment on the free and peptide amino acids in rice, wheat, and soybeans.

    PubMed

    Ahn, D J; Adeola, O; Nielsen, S S

    2001-01-01

    Controlled environments (CE) in which light, carbon dioxide, and nutrients are regulated are known to affect the chemical composition of plants. Controlled Ecological Life Support System (CELSS) environments are required for a Mars or lunar base where food resupply is both impractical and risky. Astronauts in a CELSS would need to grow and process edible biomass into foods. The complete nature of the changes in chemical composition of CE-grown plants is unknown but must be determined to ensure a safe and nutritionally adequate diet. In this article, we report the changes that occur in free and peptide-bound amino acids (AA) of select CELSS crops (rice, wheat, and soybean) grown in the field or in CE. The nonnitrate nonprotein nitrogen fraction was extracted and then analyzed for free and peptide AA. For grain or seeds, AA levels tended to increase from field to CE conditions; however, for vegetative material, AA levels remained the same or decreased from field to CE conditions. As such compositional changes are identified, researchers will be better able to design safe and nutritious diets for astronauts while minimizing needed energy and other resources.

  13. Inhibition of CD4+ T lymphocyte binding to fibronectin and immune-cell accumulation in inflammatory sites by non-peptidic mimetics of Arg-Gly-Asp.

    PubMed Central

    Hershkoviz, R; Greenspoon, N; Mekori, Y A; Hadari, R; Alon, R; Kapustina, G; Lider, O

    1994-01-01

    The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta 1 subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha 11b beta 3, and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4+ T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition. PMID:7905794

  14. D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

    PubMed

    Torres, Allan M; Tsampazi, Chryssanthi; Geraghty, Dominic P; Bansal, Paramjit S; Alewood, Paul F; Kuchel, Philip W

    2005-10-15

    The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

  15. Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

    NASA Astrophysics Data System (ADS)

    Khadsai, Sudarat; Rutnakornpituk, Boonjira; Vilaivan, Tirayut; Nakkuntod, Maliwan; Rutnakornpituk, Metha

    2016-09-01

    Magnetite nanoparticles (MNPs) were surface modified with anionic poly( N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size ( D h) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV-visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.

  16. Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

    PubMed Central

    Song, Young K.; Evangelista, Jennifer; Aschenbach, Konrad; Johansson, Peter; Wen, Xinyu; Chen, Qingrong; Lee, Albert; Hempel, Heidi; Gheeya, Jinesh S.; Getty, Stephanie; Gomez, Romel; Khan, Javed

    2013-01-01

    Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes. PMID:23593123

  17. Enduracididine, a rare amino acid component of peptide antibiotics: Natural products and synthesis

    PubMed Central

    Atkinson, Darcy J; Naysmith, Briar J; Furkert, Daniel P

    2016-01-01

    Rising resistance to current clinical antibacterial agents is an imminent threat to global public health and highlights the demand for new lead compounds for drug discovery. One such potential lead compound, the peptide antibiotic teixobactin, was recently isolated from an uncultured bacterial source, and demonstrates remarkably high potency against a wide range of resistant pathogens without apparent development of resistance. A rare amino acid residue component of teixobactin, enduracididine, is only known to occur in a small number of natural products that also possess promising antibiotic activity. This review highlights the presence of enduracididine in natural products, its biosynthesis together with a review of analogues of enduracididine. Reported synthetic approaches to the cyclic guanidine structure of enduracididine are discussed, illustrating the challenges encountered to date in the development of efficient synthetic routes to facilitate drug discovery efforts inspired by the discovery of teixobactin. PMID:28144300

  18. Stability analysis of glutamic acid linked peptides coupled to NOTA through different chemical linkages.

    PubMed

    Lang, Lixin; Ma, Ying; Kiesewetter, Dale O; Chen, Xiaoyuan

    2014-11-03

    Glutamic acid is a commonly used linker to form dimeric peptides with enhanced binding affinity than their corresponding monomeric counterparts. We have previously labeled NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 (NOTA-PRGD2) [1] with [(18)F]AlF and (68)Ga for imaging tumor angiogenesis. The p-SCN-Bn-NOTA was attached to E[c(RGDyK)]2 [2] through a mini-PEG with a thiourea linkage, and the product [1] was stable at radiolabeling condition of 100 °C and pH 4.0 acetate buffer. However, when the same p-SCN-Bn-NOTA was directly attached to the α-amine of E[c(RGDfK)]2 [3], the product NOTA-Bn-NCS-E[c(RGDfK)]2 [4] became unstable under similar conditions and the release of monomeric c(RGDfK) [5] was observed. The purpose of this work was to use HPLC and LC-MS to monitor the decomposition of glutamic acid linked dimeric peptides and their NOTA derivatives. A c(RGDyK) [6] and bombesin (BBN) [7] heterodimer c(RGDyK)-E-BBN [8], and a dimeric bombesin E(BBN)2 [9], both with a glutamic acid as the linker, along with a model compound PhSCN-E[c(RGDfK)] [10] were also studied. All the compounds were dissolved in 0.5 M pH 4.0 acetate buffer at the concentration of 1 mg/mL, and 0.1 mL of each sample was heated at 100 °C for 10 min and the more stable compounds were heated for another 30 min. The samples at both time points were analyzed with analytical HPLC to monitor the decomposition of the heated samples. The samples with decomposition were further analyzed by LC-MS to determine the mass of products from the decomposition for possible structure elucidation. After 10 min heating, the obvious release of c(RGDfK) [5] was observed for NOTA-Bn-NCS-E[c(RGDfK)]2 [4] and Ph-SCN-E[c(RGDfK)] [10]. Little or no release of monomers was observed for the remaining samples at this time point. After further heating, the release of monomers was clearly observed for E[c(RGDyK)]2 [2], E[c(RGDfK)]2 [3], c(RGDyK)-E-BBN [8], and E(BBN)2 [9]. No decomposition or little decomposition was observed for NOTA

  19. Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides

    PubMed Central

    Li, Hao; Anuwongcharoen, Nuttapat; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E. S.; Nantasenamat, Chanin

    2016-01-01

    Host defense peptides (HDPs) are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA) is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs. PMID:27376281

  20. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    NASA Astrophysics Data System (ADS)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  1. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid.

    PubMed

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A; Wepasnick, Kevin A; McDonnell, Peter; Elisseeff, Jennifer H

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  2. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  3. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  4. Tetanus toxin production is triggered by the transition from amino acid consumption to peptides.

    PubMed

    Licona-Cassani, Cuauhtemoc; Steen, Jennifer A; Zaragoza, Nicolas E; Moonen, Glenn; Moutafis, George; Hodson, Mark P; Power, John; Nielsen, Lars K; Marcellin, Esteban

    2016-10-01

    Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings.

  5. Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.

    PubMed

    Sakaguchi, Yohei; Kinumi, Tomoya; Yamazaki, Taichi; Takatsu, Akiko

    2016-02-01

    A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC-MS/MS. In this study, 1-deoxyfructosyl-VHLTPE and VHLTPE, which are N-terminal peptides of the β-chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1-bromobutane and analyzed with LC-MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non-glycated peptide.

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

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  7. Capric acid and hydroxypropylmethylcellulose increase the immunogenicity of nasally administered peptide vaccines.

    PubMed

    Nordone, Sushila K; Peacock, James W; Kirwan, Shaun M; Staats, Herman F

    2006-06-01

    Immunization by the nasal route is an established method for the induction of mucosal and systemic humoral and cell-mediated antigen-specific responses. However, the effectiveness of nasal immunization is often hampered by the need for increased doses of antigen. Bioadhesives and absorption enhancers were investigated for their ability to enhance immune responses in mice after nasal immunization with model HIV-1 peptide and protein immunogens. Two additives, hydroxypropylmethylcellulose (HPMC) and capric acid, consistently enhanced antigen-specific serum IgG endpoint titers under conditions in which antigen dose was limiting. Nasal immunization of mice with 20 microg of an HIV-1 peptide immunogen plus cholera toxin (CT) as adjuvant induced serum antipeptide IgG titers of 1:9.5log2 after four immunizations while the addition of CA or HPMC to the vaccine formulation increased serum antipeptide IgG titers to 1:15.4log2 and 1:17.6log2, respectively. When 5 microg recombinant HIV-1 gp41 was used as the immunogen, the addition of CA or HPMC to the vaccine formulation increased serum anti-gp41 IgG titers to 1:11.6log2 and 1:8.8log2, respectively, compared to 1:5.2log2 after three nasal immunizations with 5 microg gp41 + CT alone. Thus, HPMC and capric acid may be useful additives that increase the immunogenicity of nasally administered vaccines and permit less antigen to be used with each immunization.

  8. Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

    PubMed

    Camunas-Soler, Joan; Frutos, Silvia; Bizarro, Cristiano V; de Lorenzo, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramón; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2013-06-25

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.

  9. The RGD finger of Del-1 is a unique structural feature critical for integrin binding

    SciTech Connect

    Schürpf, Thomas; Chen, Qiang; Liu, Jin-huan; Wang, Rui; Springer, Timothy A.; Wang, Jia-huai

    2012-11-13

    Developmental endothelial cell locus-1 (Del-1) glycoprotein is secreted by endothelial cells and a subset of macrophages. Del-1 plays a regulatory role in vascular remodeling and functions in innate immunity through interaction with integrin {alpha}{sub V}{beta}{sub 3}. Del-1 contains 3 epidermal growth factor (EGF)-like repeats and 2 discoidin-like domains. An Arg-Gly-Asp (RGD) motif in the second EGF domain (EGF2) mediates adhesion by endothelial cells and phagocytes. We report the crystal structure of its 3 EGF domains. The RGD motif of EGF2 forms a type II' {beta} turn at the tip of a long protruding loop, dubbed the RGD finger. Whereas EGF2 and EGF3 constitute a rigid rod via an interdomain calcium ion binding site, the long linker between EGF1 and EGF2 lends considerable flexibility to EGF1. Two unique O-linked glycans and 1 N-linked glycan locate to the opposite side of EGF2 from the RGD motif. These structural features favor integrin binding of the RGD finger. Mutagenesis data confirm the importance of having the RGD motif at the tip of the RGD finger. A database search for EGF domain sequences shows that this RGD finger is likely an evolutionary insertion and unique to the EGF domain of Del-1 and its homologue milk fat globule-EGF 8. The RGD finger of Del-1 is a unique structural feature critical for integrin binding.

  10. Incorporation of extra amino acids in peptide recognition probe to improve specificity and selectivity of an electrochemical peptide-based sensor.

    PubMed

    Zaitouna, Anita J; Maben, Alex J; Lai, Rebecca Y

    2015-07-30

    We investigated the effect of incorporating extra amino acids (AA) at the n-terminus of the thiolated and methylene blue-modified peptide probe on both specificity and selectivity of an electrochemical peptide-based (E-PB) HIV sensor. The addition of a flexible (SG)3 hexapeptide is, in particular, useful in improving sensor selectivity, whereas the addition of a highly hydrophilic (EK)3 hexapeptide has shown to be effective in enhancing sensor specificity. Overall, both E-PB sensors fabricated using peptide probes with the added AA (SG-EAA and EK-EAA) showed better specificity and selectivity, especially when compared to the sensor fabricated using a peptide probe without the extra AA (EAA). For example, the selectivity factor recorded in the 50% saliva was ∼2.5 for the EAA sensor, whereas the selectivity factor was 7.8 for both the SG-EAA and EK-EAA sensors. Other sensor properties such as the limit of detection and dynamic range were minimally affected by the addition of the six AA sequence. The limit of detection was 0.5 nM for the EAA sensor and 1 nM for both SG-EAA and EK-EAA sensors. The saturation target concentration was ∼200 nM for all three sensors. Unlike previously reported E-PB HIV sensors, the peptide probe functions as both the recognition element and antifouling passivating agent; this modification eliminates the need to include an additional antifouling diluent, which simplifies the sensor design and fabrication protocol.

  11. Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications.

    PubMed

    Paulech, Jana; Liddy, Kiersten A; Engholm-Keller, Kasper; White, Melanie Y; Cordwell, Stuart J

    2015-03-01

    Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pKa Cys-SO3H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO2H/SO3H (up to 80%) from other modified peptides. We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.

  12. Observation of the side chain O-methylation of glutamic acid or aspartic acid containing model peptides by electrospray ionization-mass spectrometry.

    PubMed

    Atik, A Emin; Guray, Melda Z; Yalcin, Talat

    2017-03-15

    O-methylation of the side chains of glutamic acid (E) and aspartic acid (D) residues is generally observed modification when an acidified methanol/water (MeOH/dH2O) mixture is used as a solvent system during sample preparation for proteomic research. This chemical modification may result misidentification with endogenous protein methylation; therefore, a special care should be taken during sample handling prior to mass spectrometric analysis. In the current study, we systematically examined the extent of E/D methylation and C-terminus carboxyl group of synthetic model peptides in terms of different incubation temperatures, storage times, and added acid types as well as its percentages. To monitor these effects, C-terminus amidated and free acid forms of synthetic model peptides comprised of E or D residue(s) have been analyzed by electrospray ionization-mass spectrometry (ESI-MS). Additionally, LC-MS/MS experiments were performed to confirm the formation of methylated peptide product. The results showed that the rate of methylation was increased as the temperature increases along with prolong incubation times. Moreover, the extent of methylation was remarkably high when formic acid (FA) used as a protonation agent instead of acetic acid (AA). In addition, it was found that the degree of methylation was significantly decreased by lowering acid percentages in ESI solution. More than one acidic residue containing model peptides have been also used to explore the extent of multiple methylation reaction. Lastly, the ethanol (EtOH) and isopropanol (iPrOH) have been substituted separately with MeOH in sample preparation step to investigate the extent of esterification reaction under the same experimental conditions. However, in the positive perspective of view, this method can be used as a simple, rapid and cheap method for methylation of acidic residues under normal laboratory conditions.

  13. In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

    PubMed Central

    Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian

    2014-01-01

    To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections. PMID:24982064

  14. In vitro and in vivo activities of antimicrobial peptides developed using an amino acid-based activity prediction method.

    PubMed

    Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian; Yang, Li

    2014-09-01

    To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections.

  15. A bottom-up approach to build the hyperpolarizability of peptides and proteins from their amino acids.

    PubMed

    Duboisset, Julien; Deniset-Besseau, Ariane; Benichou, Emmanuel; Russier-Antoine, Isabelle; Lascoux, Noelle; Jonin, Christian; Hache, François; Schanne-Klein, Marie-Claire; Brevet, Pierre-François

    2013-08-29

    We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10(-30) esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10(-30) esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10(-30) esu.

  16. Bile acids induce glucagon-like peptide 2 secretion with limited effects on intestinal adaptation in early weaned pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve intestina...

  17. Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

    PubMed Central

    Liang, Wanling; Kwok, Philip C.L.; Chow, Michael Y.T.; Tang, Patricia; Mason, A. James; Chan, Hak-Kim; Lam, Jenny. K.W.

    2013-01-01

    Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impaction (NGI), gel retardation and in vitro transfection via a twin-stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation. PMID:23702276

  18. VCD studies on cyclic peptides assembled from L-α-amino acids and a trans-2-aminocyclopentane- or trans-2-aminocyclohexane carboxylic acid.

    PubMed

    Vass, E; Strijowski, U; Wollschläger, K; Mándity, I M; Szilvágyi, G; Jewgiński, M; Gaus, K; Royo, S; Majer, Z; Sewald, N; Hollósi, M

    2010-11-01

    The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans-2-aminocyclohexane carboxylic acid (Achc) or trans-2-aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three-dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6-31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR-based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides.

  19. Synthesis and Splice-Redirecting Activity of Branched, Arginine-Rich Peptide Dendrimer Conjugates of Peptide Nucleic Acid Oligonucleotides

    PubMed Central

    2010-01-01

    Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)4−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence. PMID:20879728

  20. Stability improvement of natural food colors: Impact of amino acid and peptide addition on anthocyanin stability in model beverages.

    PubMed

    Chung, Cheryl; Rojanasasithara, Thananunt; Mutilangi, William; McClements, David Julian

    2017-03-01

    Anthocyanins are prone to chemical degradation and color fading in the presence of vitamin C. The potential of three amino acids (l-phenylalanine, l-tyrosine, l-tryptophan) and a polypeptide (ε-poly-l-lysine) in prolonging the color stability of purple carrot anthocyanins (0.025%) in model beverages (0.05% l-ascorbic acid, citric acid, pH 3.0) stored at elevated temperature (40°C/7 days) was examined. In the absence of amino acids or peptides, anthocyanin degraded at first-order reaction rate. Addition of amino acids or peptide (0.1%) increased the color stability of anthocyanins, with the most significant improvement observed for l-tryptophan. The average half-life of anthocyanin color increased from 2 days to 6 days with l-tryptophan addition. Fluorescence quenching measurements revealed that the l-tryptophan interacted with anthocyanins mainly through hydrogen bonding, although some hydrophobic interaction may also have been involved. Overall, this study suggests that amino acid or peptide addition may prolong the color stability of anthocyanin in beverage products.

  1. Bone Regeneration Potential of Stem Cells Derived from Periodontal Ligament or Gingival Tissue Sources Encapsulated in RGD-Modified Alginate Scaffold

    PubMed Central

    Chen, Chider; Xu, Xingtian; Akiyama, Kentaro; Ansari, Sahar; Zadeh, Homayoun H.; Shi, Songtao

    2014-01-01

    Mesenchymal stem cells (MSCs) provide an advantageous alternative therapeutic option for bone regeneration in comparison to current treatment modalities. However, delivering MSCs to the defect site while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated bone regeneration. Here, we tested the bone regeneration capacity of periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) encapsulated in a novel RGD- (arginine-glycine-aspartic acid tripeptide) coupled alginate microencapsulation system in vitro and in vivo. Five-millimeter-diameter critical-size calvarial defects were created in immunocompromised mice and PDLSCs and GMSCs encapsulated in RGD-modified alginate microspheres were transplanted into the defect sites. New bone formation was assessed using microcomputed tomography and histological analyses 8 weeks after transplantation. Results confirmed that our microencapsulation system significantly enhanced MSC viability and osteogenic differentiation in vitro compared with non-RGD-containing alginate hydrogel microspheres with larger diameters. Results confirmed that PDLSCs were able to repair the calvarial defects by promoting the formation of mineralized tissue, while GMSCs showed significantly lower osteogenic differentiation capability. Further, results revealed that RGD-coupled alginate scaffold facilitated the differentiation of oral MSCs toward an osteoblast lineage in vitro and in vivo, as assessed by expression of osteogenic markers Runx2, ALP, and osteocalcin. In conclusion, these results for the first time demonstrated that MSCs derived from orofacial tissue encapsulated in RGD-modified alginate scaffold show promise for craniofacial bone regeneration. This treatment modality has many potential dental and orthopedic applications. PMID:24070211

  2. Nisin-induced expression of a recombinant antihypertensive peptide in dairy lactic acid bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peptides with antihypertensive activity have been identified from the enzymatic hydrolysis of bovine milk proteins. A 12-residue peptide (FFVAPFPEVFGK) shown to inhibit the angiotensin I-converting enzyme is released from the enzymatic breakdown of aS1-casein. A synthetic gene encoding this peptid...

  3. Partial d-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide

    PubMed Central

    Tugyi, Regina; Uray, Katalin; Iván, Dóra; Fellinger, Erzsébet; Perkins, Alan; Hudecz, Ferenc

    2005-01-01

    The stability of an immunogen against enzymatic degradation is considered an important factor for the design of synthetic vaccines. For our studies, we have selected an epitope from the tandem-repeat unit of the high-molecular-weight MUC2 mucin glycoprotein, which can be underglycosylated in case of colon cancer. In this study, we prepared a MUC2 peptide containing the PTGTQ epitope of a MUC2 protein backbone-specific mAb 996 and its derivatives. In these peptides, the N- and C-terminal flanking regions were systematically substituted by up to three d-amino acids. Peptides prepared by solid-phase synthesis were tested for their mAb 996 binding in competitive ELISA experiments, and their stability was studied in serum and lysosomal preparation. Our data show that the epitope function of peptide 15TPTPTGTQTPT25 is retained even in the presence of two d-amino acid residues at its N-terminal flanking region and up to three at its C-terminal flanking region (tpTPTGTQtpt). Also, this partly d peptide shows high resistance against proteolytic degradation in diluted human serum and in lysosomal preparation. These findings suggest that, by appropriate combination of structural modifications (namely, d-amino acid substitution) in the flanks of an Ab epitope, it is feasible to construct a synthetic antigen with preserved recognition properties and high stability against enzymatic degradation. Peptides tPTPTGTQTpt and tpTPTGTQTpt derived from this study can be used for immunization experiments and as potential components of synthetic vaccines for tumor therapy. PMID:15630090

  4. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  5. Progress in nanoparticulate systems for peptide, proteins and nucleic acid drug delivery.

    PubMed

    Slomkowski, Stanislaw; Gosecki, Mateusz

    2011-11-01

    Progress in many therapies, in particular in the therapies based on peptides, proteins and nucleic acids used as bioactive compounds, strongly depends on development of appropriate carriers which would be suitable for controlled delivery of the intact abovementioned compounds to required tissues, cells and intracellular compartments. This review presents last ten years' achievements and problems in development and application of synthetic polymer nanoparticulate carriers for oral, pulmonary and nasal delivery routes of oligopeptides and proteins. Whereas some traditional synthetic polymer carriers are only briefly recalled the main attention is concentrated on nanoparticles produced from functional copolymers mostly with hydroxyl, carboxyl and amino groups, suitable for immobilization of targeting moieties and for assuring prolonged circulation of nanoparticles in blood. Formulations of various nanoparticulate systems are described, including solid particles, polymer micelles, nanovesicles and nanogels, especially systems allowing drug release induced by external stimuli. Discussed are properties of these species, in particular stability in buffers and models of body fluids, loading with drugs and with drug models, drug release processes and results of biological studies. There are also discussed systems for gene delivery with special attention devoted to polymers suitable for compacting nucleic acids into nanoparticles as well as the relations between chemical structure of polymer carriers and ability of the latter for crossing cell membranes and for endosomal escape.

  6. Squaric Acid-Based Peptidic Inhibitors of Matrix Metalloprotease-1 (MMP-1)

    PubMed Central

    Onaran, M. Burak; Comeau, Anthony B.; Seto, Christopher T.

    2008-01-01

    A series of squaric acid-peptide conjugates were synthesized and evaluated as inhibitors of MMP-1. The cyclobut-3-enedione core was substituted at the 3-position with several functional groups, such as -N(alkyl)OH, -NHOH and –OH, that are designed to bind to the zinc atom in the active site of the metalloprotease. The 4-position of the cyclobut-3-enedione was derivatized with mono- or dipeptides that are designed to bind in the S1′ and S2′ subsites of the enzyme, and position the metal chelating group appropriately in the active site for binding to zinc. Positional scanning revealed that -N(Me)OH provided the highest level of inhibition among the chelating groups that were tested, and Leu-Tle-NHMe was the preferred amino acid sequence. A combination of these groups yielded an inhibitor with an IC50 value of 95 μM. For one inhibitor, conversion of one of the carbonyl groups on the cyclobut-3-enedione core to a thiocarbonyl group resulted in a 18-fold increase in potency, and yielded a compound with an IC50 value of 15 μM. PMID:16356002

  7. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    PubMed Central

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong

    2014-01-01

    Summary DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes. PMID:25246975

  8. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label.

    PubMed

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong; Wagenknecht, Hans-Achim; Vilaivan, Tirayut

    2014-01-01

    DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV-vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA-DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  9. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    NASA Astrophysics Data System (ADS)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  10. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  11. Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

    PubMed

    Patrulea, V; Hirt-Burri, N; Jeannerat, A; Applegate, L A; Ostafe, V; Jordan, O; Borchard, G

    2016-05-20

    RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

  12. Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.

    PubMed Central

    Badgujar, Shamkant B.; Mahajan, Raghunath T.

    2013-01-01

    A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing. PMID:23476742

  13. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    PubMed

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium.

  14. Assimilation of peptides and amino acids and dissimilation of lactate during submerged pure cultures of Penicillium camembertii and Geotrichum candidum.

    PubMed

    Aziza, M; Adour, L; Amrane, A

    2008-01-01

    The behavior of Penicillium camembertii and Geotrichum candidum growing in submerged pure cultures on simple (glutamate) or complex (peptones) substrates as nitrogen and carbon sources and an lactate as a second carbon source was examined. Similar to the behavior previously recorded on a simple substrate (glutamate), a clear differentiation between the carbon source and the energy source was also shown on peptones and lactate during P. camembertii growth, since throughout growth, lactate was only dissimilated, viz., used for energy supply by oxidation into CO2, whereas peptides and amino acids from peptones were used for carbon (and nitrogen) assimilation. Because of its deaminating activity, G. candidum preferred peptides and amino acids to lactate as energy sources, in addition to being assimilated as carbon and nitrogen sources. From this, on peptones and lactate, G. candidum grew faster than P. camembertii (0.19 and 0.08 g/l/h, respectively) by assimilating the most readily utilizable peptides and amino acids; however, owing to its lower proteolytic activity, the maximum biomass was lower than that of P. camembertii (3.7 and 5.5 g/l, respectively), for which continuous proteolysis and assimilation of peptides were shown.

  15. Unraveling the Mechanisms of Peptide-Mediated Delivery of Nucleic Acids Using Electron Microscopy.

    PubMed

    Margus, Helerin; Juks, Carmen; Pooga, Margus

    2015-01-01

    Cell-penetrating peptides (CPPs) are efficient non-viral delivery vectors for bioactive cargos, both in vitro and in vivo. Cargo molecules can be attached to CPPs either via covalent conjugation or by complex formation using co-incubation, which is typically used for charged molecules such as nucleic acids. The latter technique is efficiently used in case of CADY, MPG, Pep peptides, NickFects and PepFects that condense oligonucleotides (ONs) into nanoparticles, which efficiently enter cells and induce biological effects. Despite being highly promising candidates for developing new-generation medicines, CPPs' internalization mechanisms and intracellular trafficking are still far from being well-understood, and obtained data are often controversial. Transmission electron microscopy (TEM) is an informative and valuable tool for examining the mechanisms of CPP-ON nanoparticles. TEM enables to visualize nanoparticles or single molecules labeled with Nanogold™ tag, and follow their association with cells and intracellular localization. In this chapter, we present methods for preparation of CPP-ON nanoparticles for TEM analysis and for examination of their interactions with the plasma membrane, and subsequent cellular uptake either by direct translocation or endocytosis. In case of endocytosis, ONs have to be released from endosomes and reach their target site in nucleus or cytoplasm to reveal their activity. TEM enables to estimate when the endosomal escape begins, from which type of endosomal vesicles it occurs, whether the vesicles are broken, or nanocomplexes translocate across the membrane into cytosol. Since single ONs could be followed, the time-frame that is necessary for the splice-switching nucleotides to translocate into cell nucleus can be analyzed by TEM.

  16. Helix 69 of E. coli 23S ribosomal RNA as a peptide nucleic acid target.

    PubMed

    Kulik, Marta; Markowska-Zagrajek, Agnieszka; Wojciechowska, Monika; Grzela, Renata; Wituła, Tomasz; Trylska, Joanna

    2017-04-07

    A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines () and U1915 with 3-methyl-. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA. We also verified the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which turned out to be in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.

  17. Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

    PubMed Central

    Cordier, Céline; Boutimah, Fatima; Bourdeloux, Mathilde; Dupuy, Florian; Met, Elisabeth; Alberti, Patrizia; Loll, François; Chassaing, Gérard; Burlina, Fabienne; Saison-Behmoaras, Tula Ester

    2014-01-01

    Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization. PMID:25127364

  18. Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA.

    PubMed

    Takahashi, T; Hamasaki, K; Ueno, A; Mihara, H

    2001-04-01

    In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.

  19. Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

    PubMed

    Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

    2016-03-15

    Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.

  20. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

    PubMed Central

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

    2017-01-01

    Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

  1. Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. II. Amino acid sequence of the tryptic peptides.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1980-04-10

    Amino acid sequence studies of tryptic peptides isolated from a histidine-rich fragment (Cp F5) of human ceruloplasmin are described. Nineteen tryptic peptides were isolated from unmodified Cp F5 and five tryptic peptides were isolated from citraconylated Cp F5. These peptides, together with the cyanogen bromide fragments reported previously, allowed the assembly of the complete sequence of Cp F5. The fragment has 159 residues and a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains 1 free cysteine that may be part of a copper-binding site. Human ceruloplasmin is a single polypeptide chain with a molecular weight of about 130,000 that is readily cleaved to large fragments by proteolytic enzymes; the relationships of Cp F5 to intact ceruloplasmin and to structural subunits earlier proposed is described. Cp F5 probably is an intact globular domain that is attached to the COOH-terminal end of ceruloplasmin by a labile interdomain peptide bond.

  2. Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide

    PubMed Central

    Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori

    2016-01-01

    The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20–500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection. PMID:27457924

  3. Targeting In-Stent-Stenosis with RGD- and CXCL1-Coated Mini-Stents in Mice

    PubMed Central

    Weinandy, Stefan; Schreiber, Fabian; Megens, Remco T. A.; Theelen, Wendy; Smeets, Ralf; Jockenhövel, Stefan; Gries, Thomas; Möller, Martin; Klee, Doris; Weber, Christian; Zernecke, Alma

    2016-01-01

    Atherosclerotic lesions that critically narrow the artery can necessitate an angioplasty and stent implantation. Long-term therapeutic effects, however, are limited by excessive arterial remodeling. We here employed a miniaturized nitinol-stent coated with star-shaped polyethylenglycole (star-PEG), and evaluated its bio-functionalization with RGD and CXCL1 for improving in-stent stenosis after implantation into carotid arteries of mice. Nitinol foils or stents (bare metal) were coated with star-PEG, and bio-functionalized with RGD, or RGD/CXCL1. Cell adhesion to star-PEG-coated nitinol foils was unaltered or reduced, whereas bio-functionalization with RGD but foremost RGD/CXCL1 increased adhesion of early angiogenic outgrowth cells (EOCs) and endothelial cells but not smooth muscle cells when compared with bare metal foils. Stimulation of cells with RGD/CXCL1 furthermore increased the proliferation of EOCs. In vivo, bio-functionalization with RGD/CXCL1 significantly reduced neointima formation and thrombus formation, and increased re-endothelialization in apoE-/- carotid arteries compared with bare-metal nitinol stents, star-PEG-coated stents, and stents bio-functionalized with RGD only. Bio-functionalization of star-PEG-coated nitinol-stents with RGD/CXCL1 reduced in-stent neointima formation. By supporting the adhesion and proliferation of endothelial progenitor cells, RGD/CXCL1 coating of stents may help to accelerate endothelial repair after stent implantation, and thus may harbor the potential to limit the complication of in-stent restenosis in clinical approaches. PMID:27192172

  4. RGD-based active targeting of novel polycation liposomes bearing siRNA for cancer treatment.

    PubMed

    Yonenaga, Norihito; Kenjo, Eriya; Asai, Tomohiro; Tsuruta, Atsushi; Shimizu, Kosuke; Dewa, Takehisa; Nango, Mamoru; Oku, Naoto

    2012-06-10

    For the purpose of systemic delivery of siRNA, we previously developed polycation liposomes (PCLs) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as an effective siRNA carrier. In the present study, to endow these PCLs (TEPA-PCL) actively target cancer cells and angiogenic vessels, we decorated the PCLs with cyclic RGD, by using cyclic RGD-grafted distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and investigated the usefulness of this type of carrier (RGD-PEG-PCL) for active targeting. Firstly, the gene-silencing efficacy of siRNA for luciferase (siLuc2) formulated in RGD-PEG-PCL (RGD-PEG-PCL/siLuc2) was examined in vitro by using B16F10-luc2 murine melanoma cells stably expressing the luciferase 2 gene, where the siRNA was grafted with cholesterol at the 3'-end of the sense strand (siRNA-C) for the stable association of the siRNA with the PCL. RGD-PEG-PCL/siLuc2 showed high knockdown efficiency compared with siLuc2 formulated in PEGylated TEPA-PCL without cyclic RGD (PEG-PCL). Next, the gene-silencing efficacy of RGD-PEG-PCL/siLuc2 was examined in vivo by use of B16F10-luc2 lung metastatic model mice. The intravenous injection of RGD-PEG-PCL/siLuc2 showed high knockdown efficiency against metastatic B16F10-luc2 tumors in the lungs of the mice, as assessed with an in vivo imaging system. These data strongly suggest that systemic and active targeting siRNA delivery using RGD-PEG-PCL is useful for cancer RNAi therapy.

  5. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins.

    PubMed

    Koehbach, Johannes; Gruber, Christian W; Becker, Christian; Kreil, David P; Jilek, Alexander

    2016-05-06

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods.

  6. Killing of Mycobacterium avium by Lactoferricin Peptides: Improved Activity of Arginine- and d-Amino-Acid-Containing Molecules

    PubMed Central

    Silva, Tânia; Magalhães, Bárbara; Maia, Sílvia; Gomes, Paula; Nazmi, Kamran; Bolscher, Jan G. M.; Rodrigues, Pedro N.; Bastos, Margarida

    2014-01-01

    Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections. PMID:24709266

  7. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins

    PubMed Central

    2016-01-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  8. Analysis of peptide-protein binding using amino acid descriptors: prediction and experimental verification for human histocompatibility complex HLA-A0201.

    PubMed

    Guan, Pingping; Doytchinova, Irini A; Walshe, Valerie A; Borrow, Persephone; Flower, Darren R

    2005-11-17

    Amino acid descriptors are often used in quantitative structure-activity relationship (QSAR) analysis of proteins and peptides. In the present study, descriptors were used to characterize peptides binding to the human MHC allele HLA-A0201. Two sets of amino acid descriptors were chosen: 93 descriptors taken from the amino acid descriptor database AAindex and the z descriptors defined by Wold and Sandberg. Variable selection techniques (SIMCA, genetic algorithm, and GOLPE) were applied to remove redundant descriptors. Our results indicate that QSAR models generated using five z descriptors had the highest predictivity and explained variance (q2 between 0.6 and 0.7 and r2 between 0.6 and 0.9). Further to the QSAR analysis, 15 peptides were synthesized and tested using a T2 stabilization assay. All peptides bound to HLA-A0201 well, and four peptides were identified as high-affinity binders.

  9. Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

    NASA Technical Reports Server (NTRS)

    Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

    1998-01-01

    The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

  10. Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

    PubMed

    Ferreira, André M; Cruz-Moreira, Daniela; Cerqueira, Laura; Miranda, João M; Azevedo, Nuno F

    2017-03-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.

  11. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  12. A peptide nucleic acid targeting nuclear RAD51 sensitizes multiple myeloma cells to melphalan treatment

    PubMed Central

    Alagpulinsa, David Abasiwani; Yaccoby, Shmuel; Ayyadevara, Srinivas; Shmookler Reis, Robert Joseph

    2015-01-01

    RAD51-mediated recombinational repair is elevated in multiple myeloma (MM) and predicts poor prognosis. RAD51 has been targeted to selectively sensitize and/or kill tumor cells. Here, we employed a peptide nucleic acid (PNA) to inhibit RAD51 expression in MM cells. We constructed a PNA complementary to a unique segment of the RAD51 gene promoter, spanning the transcription start site, and conjugated it to a nuclear localization signal (PKKKRKV) to enhance cellular uptake and nuclear delivery without transfection reagents. This synthetic construct, (PNArad51_nls), significantly reduced RAD51 transcripts in MM cells, and markedly reduced the number and intensity of de novo and melphalan-induced nuclear RAD51 foci, while increasing the level of melphalan-induced γH2AX foci. Melphalan alone markedly induced the expression of 5 other genes involved in homologous-recombination repair, yet suppression of RAD51 by PNArad51_nls was sufficient to synergize with melphalan, producing significant synthetic lethality of MM cells in vitro. In a SCID-rab mouse model mimicking the MM bone marrow microenvironment, treatment with PNArad51_nls ± melphalan significantly suppressed tumor growth after 2 weeks, whereas melphalan plus control PNArad4µ_nls was ineffectual. This study highlights the importance of RAD51 in myeloma growth and is the first to demonstrate that anti-RAD51 PNA can potentiate conventional MM chemotherapy. PMID:25996477

  13. Amyloid-β peptide (1-42) aggregation induced by copper ions under acidic conditions.

    PubMed

    Bin, Yannan; Li, Xia; He, Yonghui; Chen, Shu; Xiang, Juan

    2013-07-01

    It is well known that the aggregation of amyloid-β peptide (Aβ) induced by Cu²⁺ is related to incubation time, solution pH, and temperature. In this work, the aggregation of Aβ₁₋₄₂ in the presence of Cu²⁺ under acidic conditions was studied at different incubation time and temperature (e.g. 25 and 37°C). Incubation temperature, pH, and the presence of Cu²⁺ in Aβ solution were confirmed to alter the morphology of aggregation (fibrils or amorphous aggregates), and the morphology is pivotal for Aβ neurotoxicity and Alzheimer disease (AD) development. The results of atomic force microscopy (AFM) indicated that the formation of Aβ fibrous morphology is preferred at lower pH, but Cu²⁺ induced the formation of amorphous aggregates. The aggregation rate of Aβ was increased with the elevation of temperature. These results were further confirmed by fluorescence spectroscopy and circular dichroism spectroscopy and it was found that the formation of β-sheet structure was inhibited by Cu²⁺ binding to Aβ. The result was consistent with AFM observation and the fibrillation process was restrained. We believe that the local charge state in hydrophilic domain of Aβ may play a dominant role in the aggregate morphology due to the strong steric hindrance. This research will be valuable for understanding of Aβ toxicity in AD.

  14. Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid

    PubMed Central

    Layek, Buddhadev; Lipp, Lindsey; Singh, Jagdish

    2015-01-01

    Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs) for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA. PMID:26690119

  15. Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism

    PubMed Central

    Hong, Christopher S; Yang, Chunzhang; Zhuang, Zhengping

    2016-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with many applications. Compared with DNA, PNAs bind their complementary DNA strand with higher specificity and strength, an attribute that can make it an effective polymerase chain reaction clamp. A growing body of work has demonstrated the utility of PNAs in detecting low levels of mutant DNA, particularly in the detection of circulating mutated tumor cells in the peripheral blood. The PNA-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid than next-generation deep sequencing. We have previously demonstrated that PNAs can successfully detect somatic mosaicism in patients with suspected disease phenotypes. In this report, we detail our methodology behind PNA design and application. We describe our protocol for optimizing the PNA for sequencing use and for determining the sensitivity of the PNA-based assay. Lastly, we discuss the potential applications of our assay for future laboratory and clinical purposes and highlight the role of PNAs in the detection of somatic mosaicism. PMID:27115839

  16. Intracellular delivery of peptide nucleic acid and organic molecules using zeolite-L nanocrystals.

    PubMed

    Bertucci, Alessandro; Lülf, Henning; Septiadi, Dedy; Manicardi, Alex; Corradini, Roberto; De Cola, Luisa

    2014-11-01

    The design and synthesis of smart nanomaterials can provide interesting potential applications for biomedical purposes from bioimaging to drug delivery. Manufacturing multifunctional systems in a way to carry bioactive molecules, like peptide nucleic acids able to recognize specific targets in living cells, represents an achievement towards the development of highly selective tools for both diagnosis and therapeutics. This work describes a very first example of the use of zeolite nanocrystals as multifunctional nanocarriers to deliver simultaneously PNA and organic molecules into living cells. Zeolite-L nanocrystals are functionalized by covalently attaching the PNA probes onto the surface, while the channel system is filled with fluorescent guest molecules. The cellular uptake of the PNA/Zeolite-L hybrid material is then significantly increased by coating the whole system with a thin layer of biodegradable poly-L-lysine. The delivery of DAPI as a model drug molecule, inserted into the zeolite pores, is also demonstrated to occur in the cells, proving the multifunctional ability of the system. Using this zeolite nanosystem carrying PNA probes designed to target specific RNA sequences of interest in living cells could open new possibilities for theranostic and gene therapy applications.

  17. Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons.

    PubMed

    Li, Xu; Morgenroth, Eberhard; Raskin, Lutgarde

    2008-12-01

    The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

  18. Efficient inhibition of miR-155 function in vivo by peptide nucleic acids

    PubMed Central

    Fabani, Martin M.; Abreu-Goodger, Cei; Williams, Donna; Lyons, Paul A.; Torres, Adrian G.; Smith, Kenneth G. C.; Enright, Anton J.; Gait, Michael J.; Vigorito, Elena

    2010-01-01

    MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use. PMID:20223773

  19. Human leukocyte antigen haplotype phasing by allele-specific enrichment with peptide nucleic acid probes

    PubMed Central

    Murphy, Nicholas M; Pouton, Colin W; Irving, Helen R

    2014-01-01

    Targeted capture of large fragments of genomic DNA that enrich for human leukocyte antigen (HLA) system haplotypes has utility in haematopoietic stem cell transplantation. Current methods of HLA matching are based on inference or familial studies of inheritance; and each approach has its own inherent limitations. We have designed and tested a probe–target-extraction method for capturing specific HLA haplotypes by hybridization of peptide nucleic acid (PNA) probes to alleles of the HLA-DRB1 gene. Short target fragments contained in plasmids were initially used to optimize the method followed by testing samples of genomic DNA from human subjects with preselected HLA haplotypes and obtained approximately 10% enrichment for the specific haplotype. When performed with high-molecular-weight genomic DNA, 99.0% versus 84.0% alignment match was obtained for the specific haplotype probed. The allele-specific target enrichment that we obtained can facilitate the elucidation of haplotypes between the 65 kb separating the HLA-DRB1 and the HLA-DQA1 genes, potentially spanning a total distance of at least 130 kb. Allele-specific target enrichment with PNA probes is a straightforward technique that has the capability to improve the resolution of DNA and whole genome sequencing technologies by allowing haplotyping of enriched DNA and crucially, retaining the DNA methylation profile. PMID:24936514

  20. DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

    PubMed Central

    Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

    2009-01-01

    The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that the polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence-specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry, but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step towards the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each

  1. Electrochemical paper-based peptide nucleic acid biosensor for detecting human papillomavirus.

    PubMed

    Teengam, Prinjaporn; Siangproh, Weena; Tuantranont, Adisorn; Henry, Charles S; Vilaivan, Tirayut; Chailapakul, Orawon

    2017-02-01

    A novel paper-based electrochemical biosensor was developed using an anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) and graphene-polyaniline (G-PANI) modified electrode to detect human papillomavirus (HPV). An inkjet printing technique was employed to prepare the paper-based G-PANI-modified working electrode. The AQ-PNA probe baring a negatively charged amino acid at the N-terminus was immobilized onto the electrode surface through electrostatic attraction. Electrochemical impedance spectroscopy (EIS) was used to verify the AQ-PNA immobilization. The paper-based electrochemical DNA biosensor was used to detect a synthetic 14-base oligonucleotide target with a sequence corresponding to human papillomavirus (HPV) type 16 DNA by measuring the electrochemical signal response of the AQ label using square-wave voltammetry before and after hybridization. It was determined that the current signal significantly decreased after the addition of target DNA. This phenomenon is explained by the rigidity of PNA-DNA duplexes, which obstructs the accessibility of electron transfer from the AQ label to the electrode surface. Under optimal conditions, the detection limit of HPV type 16 DNA was found to be 2.3 nM with a linear range of 10-200 nM. The performance of this biosensor on real DNA samples was tested with the detection of PCR-amplified DNA samples from the SiHa cell line. The new method employs an inexpensive and disposable device, which easily incinerated after use and is promising for the screening and monitoring of the amount of HPV-DNA type 16 to identify the primary stages of cervical cancer.

  2. RGD Surface Functionalization of the Hydrophilic Acrylic Intraocular Lens Material to Control Posterior Capsular Opacification

    PubMed Central

    Huang, Yi-Shiang; Bertrand, Virginie; Bozukova, Dimitriya; Pagnoulle, Christophe; Labrugère, Christine; De Pauw, Edwin; De Pauw-Gillet, Marie-Claire; Durrieu, Marie-Christine

    2014-01-01

    Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient's age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development. PMID:25501012

  3. Intravascularly Administered RGD-Displaying Measles Viruses Bind to and Infect Neovessel Endothelial Cells In Vivo

    PubMed Central

    Ong, Hooi Tin; Trejo, Theodore R; Pham, Linh D; Oberg, Ann L; Russell, Stephen J; Peng, Kah-Whye

    2009-01-01

    Systemically administered vectors must cross the endothelial lining of tumor blood vessels to access cancer cells. Vectors that interact with markers on the lumenal surface of these endothelial cells might have enhanced tumor localization. Here, we generated oncolytic measles viruses (MVs) displaying αvβ3 integrin-binding peptides, cyclic arginine-glycine-aspartate (RGD) or echistatin, on the measles hemagglutinin protein. Both viruses had expanded tropisms, and efficiently entered target cells via binding to integrins, but also retained their native tropisms for CD46 and signaling lymphocyte activation molecule (SLAM). When fluorescently labeled and injected intravascularly into chick chorioallantoic membranes (CAMs), in contrast to unmodified viruses, the integrin-binding viral particles bound to the lumenal surface of the developing chick neovessels and infected the CAM vascular endothelial cells. In a mouse model of VEGF-induced angiogenesis in the ear pinna, the integrin-binding viruses, but not the parental virus, infected cells at sites of new blood vessel formation. When given intravenously to mice bearing tumor xenografts, the integrin-binding virus infected endothelial cells of tumor neovessels in addition to tumor parenchyma. To our knowledge, this is the first report demonstrating that oncolytic MVs can be engineered to target the lumenal endothelial surface of newly formed blood vessels when administered intravenously in living animals. PMID:19277014

  4. [A new SVRDF 3D-descriptor of amino acids and its application to peptide quantitative structure activity relationship].

    PubMed

    Tong, Jian-Bo; Zhang, Sheng-Wan; Cheng, Su-Li; Li, Gai-Xian

    2007-01-01

    To establish a new amino acid structure descriptor that can be applied to polypeptide quantitative structure activity relationship (QSAR) studies, a new descriptor, SVRDF, was derived from a principal components analysis of a matrix of 150 radial distribution function index of amino acids. The scale was then applied in three panels of peptide QSAR that were molded by partial least squares regression. The obtained models with the correlation coefficients (R2(cum)), cross-validation correlation coefficients (Q2(cum)) were 0.766 and 0.724 for 48 bitter tasting dipeptides; 0.941 and 0.811 for 21 oxytocin analogues; 0.996 and 0.919 for 20 thromboplastin inhibitors. Satisfactory results showed that information related to biological activity can be systemically expressed by SVRDF scales, which may be an useful structural expression methodology for the study of peptides QSAR.

  5. Localized surface plasmon resonance interfaces coated with poly[3-(pyrrolyl)carboxylic acid] for histidine-tagged peptide sensing.

    PubMed

    Tighilt, Fatma-Zohra; Subramanian, Palaniappan; Belhaneche-Bensemra, Naima; Boukherroub, Rabah; Gabouze, Noureddine; Sam, Sabrina; Szunerits, Sabine

    2011-10-21

    The paper reports on a novel localized surface plasmon resonance (LSPR) substrate architecture for the immobilization and detection of histidine-tagged peptides. The LSPR interface consists of an ITO (indium tin oxide) substrate coated with gold nanostructures. The latter are obtained by thermal deposition of a thin (2 nm thick) gold film followed by post-annealing at 500 °C. The LSPR interface was coated with poly[3-(pyrrolyl)carboxylic acid] thin films using electrochemical means. The ability of the LSPR interfaces coated with poly[3-(pyrrolyl)carboxylic acid] to chelate copper ions was investigated. Once loaded with metal ions, the modified LSPR interface was able to bind specifically to histidine-tagged peptides. The binding process was followed using LSPR.

  6. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

    PubMed

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X; Huang, Guowei

    2015-10-20

    Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production.

  7. Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An eight amino acid fragment (PFPEVFGK) of a known milk protein-derived antihypertensive peptide was synthesized by microwave-assisted solid phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit the angiotensin-converting enzyme was assessed and compared to that of the ...

  8. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    SciTech Connect

    Garrison, W. M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  9. Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies.

    PubMed

    Beavers, Kelsey R; Werfel, Thomas A; Shen, Tianwei; Kavanaugh, Taylor E; Kilchrist, Kameron V; Mares, Jeremy W; Fain, Joshua S; Wiese, Carrie B; Vickers, Kasey C; Weiss, Sharon M; Duvall, Craig L

    2016-09-01

    Self-assembled polymer/porous silicon nanocomposites overcome intracellular and systemic barriers for in vivo application of peptide nucleic acid (PNA) anti-microRNA therapeutics. Porous silicon (PSi) is leveraged as a biodegradable scaffold with high drug-cargo-loading capacity. Functionalization with a diblock polymer improves PSi nanoparticle colloidal stability, in vivo pharmacokinetics, and intracellular bioavailability through endosomal escape, enabling PNA to inhibit miR-122 in vivo.

  10. Tri-peptide reference structures for the calculation of relative solvent accessible surface area in protein amino acid residues.

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2015-02-01

    Relative amino acid residue solvent accessibility values allow the quantitative comparison of atomic solvent-accessible surface areas in different residue types and physical environments in proteins and in protein structural alignments. Geometry-optimised tri-peptide structures in extended solvent-exposed reference conformations have been obtained for 43 amino acid residue types at a high level of quantum chemical theory. Significant increases in side-chain solvent accessibility, offset by reductions in main-chain atom solvent exposure, were observed for standard residue types in partially geometry-optimised structures when compared to non-minimised models built from identical sets of proper dihedral angles abstracted from the literature. Optimisation of proper dihedral angles led most notably to marked increases of up to 54% in proline main-chain atom solvent accessibility compared to literature values. Similar effects were observed for fully-optimised tri-peptides in implicit solvent. The relief of internal strain energy was associated with systematic variation in N, C(α) and C(β) atom solvent accessibility across all standard residue types. The results underline the importance of optimisation of 'hard' degrees of freedom (bond lengths and valence bond angles) and improper dihedral angle values from force field or other context-independent reference values, and impact on the use of standardised fixed internal co-ordinate geometry in sampling approaches to the determination of absolute values of protein amino acid residue solvent accessibility. Quantum chemical methods provide a useful and accurate alternative to molecular mechanics methods to perform energy minimisation of peptides containing non-standard (chemically modified) amino acid residues frequently present in experimental protein structure data sets, for which force field parameters may not be available. Reference tri-peptide atomic co-ordinate sets including hydrogen atoms are made freely available.

  11. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    PubMed

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH.

  12. Evaluation of the Influence of Amino Acid Composition on the Propensity for Collision-Induced Dissociation of Model Peptides Using Molecular Dynamics Simulations

    SciTech Connect

    Cannon, William R.; Taasevigen, Danny J.; Baxter, Douglas J.; Laskin, Julia

    2007-09-01

    The dynamical behavior of model peptides was evaluated with respect to their ability to form internal proton donor-acceptor pairs using molecular dynamics simulations. The proton donor-acceptor pairs are postulated to be prerequisites for peptide bond cleavage resulting in formation of b and y ions during low energy collision-induced dissociation in tandem mass spectrometry (MS/MS). The simulations for the polyalanine pentamer Ala5H+ were compared to experimental data from collision energy-resolved surface induced dissociation (SID) studies. The results of the simulation are insightful into the events that likely lead up to the fragmentation of peptides. 9-mer polyalanine-based model peptides were used to examine the dynamical effect of each of the 20 common amino acids on the probability to form donor-acceptor pairs at labile peptide bonds. A continuous range of probabilities was observed as a function of the substituted amino acid. However, the location of the peptide bond involved in the donor-acceptor pair plays a critical role in the dynamical behavior. This influence of position on the probability of forming a donor-acceptor pair would be hard to predict from statistical analyses on experimental spectra of aggregate, diverse peptides. In addition, the inclusion of basic side chains in the model peptides alters the probability of forming donor-acceptor pairs across the entire backbone. In this case there are still more ionizing protons than basic residues, but the side chains of the basic amino acids form stable hydrogen bond networks with the peptide carbonyl oxygens and thus act to prevent free access of “mobile protons” to labile peptide bonds. It is clear from the work that the identification of peptides from low-energy CID using automated computational methods should consider the location of the fragmenting bond as well as the amino acid composition.

  13. Synthetic amphibian peptides and short amino-acids derivatives against planktonic cells and mature biofilm of Providencia stuartii clinical strains.

    PubMed

    Ostrowska, Kinga; Kamysz, Wojciech; Dawgul, Małgorzata; Różalski, Antoni

    2014-01-01

    Over the last decade, the growing number of multidrug resistant strains limits the use of many of the currently available chemotherapeutic agents. Furthermore, bacterial biofilm, due to its complex structure, constitutes an effective barrier to conventional antibiotics. The in vitro activities of naturally occurring peptide (Citropin 1.1), chemically engineered analogue (Pexiganan), newly-designed, short amino-acid derivatives (Pal-KK-NH2, Pal-KKK-NH2, Pal-RRR-NH2) and six clinically used antimicrobial agents (Gatifloxacin, Ampicilin, Cefotaxime, Ceftriaxone, Cefuroxime and Cefalexin) were investigated against planktonic cells and mature biofilm of multidrug-resistant Providencia stuartii strains, isolated from urological catheters. The MICs, MBCs values were determined by broth microdilution technique. Inhibition of biofilm formation by antimicrobial agents as well as biofilm susceptibility assay were tested using a surrogate model based on the Crystal Violet method. The antimicrobial activity of amino-acids derivatives and synthetic peptides was compared to that of clinically used antibiotics. For planktonic cells, MICs of peptides and antibiotics ranged between 1 and 256 μg/ml and 256 and ≥ 2048 μg/ml, respectively. The MBCs values of Pexiganan, Citropin 1.1 and amino-acids derivatives were between 16 and 256 μg/ml, 64 and 256 μg/ml and 16 and 512 μg/ml, respectively. For clinically used antibiotics the MBCs values were above 2048 μg/ml. All of the tested peptides and amino-acids derivatives, showed inhibitory activity against P. stuartii biofilm formation, in relation to their concentrations. Pexiganan and Citropin 1.1 in concentration range 32 and 256 μg/ml caused both strong and complete suppression of biofilm formation. None of the antibiotics caused complete inhibition of biofilm formation process. The biofilm susceptibility assay verified the extremely poor antibiofilm activity of conventional antibiotics compared to synthetic peptides. The

  14. Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

    PubMed Central

    Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

    2010-01-01

    This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

  15. Human immunodeficiency virus trans-activator of transcription peptide detection via ribonucleic acid aptamer on aminated diamond biosensor

    NASA Astrophysics Data System (ADS)

    Rahim Ruslinda, A.; Wang, Xianfen; Ishii, Yoko; Ishiyama, Yuichiro; Tanabe, Kyosuke; Kawarada, Hiroshi

    2011-09-01

    The potential of ribonucleic acid (RNA) as both informational and ligand binding molecule have opened a scenario in the development of biosensors. An aminated diamond-based RNA aptasensor is presented for human immunodeficiency virus (HIV) trans-activator of transcription (Tat) peptide protein detection that not only gives a labeled or label-free detection method but also provides a reusable platform for a simple, sensitive, and selective detection of proteins. The immobilized procedure was based on the binding interaction between positively charged amine terminated diamond and the RNA aptamer probe molecules with the negatively charged surface carboxylic compound linker molecule such as terephthalic acid.

  16. Influence of peptides and amino acids on fermentation rate and de novo synthesis of amino acids by mixed micro-organisms from the sheep rumen.

    PubMed

    Atasoglu, C; Valdés, C; Newbold, C J; Wallace, R J

    1999-04-01

    The influence of different N sources on fermentation rate and de novo amino acid synthesis by rumen micro-organisms was investigated in vitro using rumen fluid taken from four sheep receiving a mixed diet comprising (g/kg DM): grass hay 500, barley 299.5, molasses 100, fish meal 91, minerals and vitamins 9.5. Pancreatic casein hydrolysate (P; comprising mainly peptides with some free amino acids; 10 g/l), free amino acids (AA; casein acid hydrolysate + added cysteine and tryptophan; 10 g/l), or a mixture of L-proline, glycine, L-valine and L-threonine (M; 0.83 g/l each) were added to diluted (1:3, v/v), strained rumen fluid along with 15NH4Cl (A; 1.33 g/l) and 6.7 g/l of a mixture of starch, cellobiose and xylose (1:1:1, by weight). P and AA, but not M, stimulated net gas production after 4 and 8 h incubation (P < 0.05) in comparison with A alone. P increased microbial-protein synthesis (P < 0.05) compared with the other treatments. All of the microbial-N formed after 10 h was synthesized de novo from 15NH3 in treatment A, and the addition of pre-formed amino acids decreased the proportion to 0.37, 0.55, and 0.86 for P, AA, and M respectively. De novo synthesis of amino acids (0.29, 0.42 and 0.69 respectively) was lower than cell-N. Enrichment of alanine, glutamate and aspartate was slightly higher than that of other amino acids, while enrichment in proline was much lower, such that 0.83-0.95 of all proline incorporated into particulate matter was derived from pre-formed proline. Glycine, methionine, lysine, valine and threonine tended to be less enriched than other amino acids. The form in which the amino acids were supplied, as P or AA, had little influence on the pattern of de novo synthesis. When the concentration of peptides was decreased, the proportion of microbial-N formed from NH3 increased, so that at an initial concentration of 1 g peptides/l, similar to the highest reported ruminal peptide concentrations, 0.68 of cell-N was formed from NH3. Decreasing

  17. Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

    NASA Astrophysics Data System (ADS)

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-12-01

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

  18. Evolutionary importance of the intramolecular pathways of hydrolysis of phosphate ester mixed anhydrides with amino acids and peptides.

    PubMed

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-12-11

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

  19. Development of helix-stabilized cell-penetrating peptides containing cationic α,α-disubstituted amino acids as helical promoters.

    PubMed

    Yamashita, Hiroko; Misawa, Takashi; Oba, Makoto; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2017-03-15

    Cell-penetrating peptides (CPP) have attracted many scientists' attention as intracellular delivery tools due to their high cargo molecule transportation efficiency and low cytotoxicity. Therefore, in many research fields CPP, such as HIV-Tat and oligoarginine (Rn), are used to deliver hydrophilic drugs and biomolecules, including proteins, DNA, and RNA. We designed four types of CPP that contained cationic α,α-disubstituted amino acids (Api(C2Gu) and Api(C4Gu)) as helical promoters; i.e., 1-4 [FAM-β-Ala-(l-Arg-l-Arg-Xaa)3-(Gly)3-NH2 (1: Xaa=Api(C2Gu), 2: Xaa=Api(C4Gu)), 3: FAM-β-Ala-(l-Arg)8-Api(C2Gu)-(Gly)3-NH2, and 4: FAM-β-Ala-(l-Arg)5-Api(C2Gu)-(l-Arg)2-Api(C2Gu)-(Gly)3-NH2], and investigated their preferred secondary structures and cell membrane-penetrating ability. As a result, we found that the permeation efficiency of the CPP was affected by the number of helical promoters in their sequences. Specially, peptide 1, which contained three Api(C2Gu) residues, formed a stable helical structure and passed through the cell membrane more efficiently than the other peptides. Moreover, it was demonstrated that the spatial arrangement of the peptides' side chains also influenced their permeability and the helical stabilization of their main chains.

  20. Peptide Conformer Acidity Analysis of Protein Flexibility Monitored by Hydrogen Exchange†

    PubMed Central

    2009-01-01

    The amide hydrogens that are exposed to solvent in the high-resolution X-ray structures of ubiquitin, FK506-binding protein, chymotrypsin inhibitor 2, and rubredoxin span a billion-fold range in hydroxide-catalyzed exchange rates which are predictable by continuum dielectric methods. To facilitate analysis of transiently accessible amides, the hydroxide-catalyzed rate constants for every backbone amide of ubiquitin were determined under near physiological conditions. With the previously reported NMR-restrained molecular dynamics ensembles of ubiquitin (PDB codes 2NR2 and 2K39) used as representations of the Boltzmann-weighted conformational distribution, nearly all of the exchange rates for the highly exposed amides were more accurately predicted than by use of the high-resolution X-ray structure. More strikingly, predictions for the amide hydrogens of the NMR relaxation-restrained ensemble that become exposed to solvent in more than one but less than half of the 144 protein conformations in this ensemble were almost as accurate. In marked contrast, the exchange rates for many of the analogous amides in the residual dipolar coupling-restrained ubiquitin ensemble are substantially overestimated, as was particularly evident for the Ile 44 to Lys 48 segment which constitutes the primary interaction site for the proteasome targeting enzymes involved in polyubiquitylation. For both ensembles, “excited state” conformers in this active site region having markedly elevated peptide acidities are represented at a population level that is 102 to 103 above what can exist in the Boltzmann distribution of protein conformations. These results indicate how a chemically consistent interpretation of amide hydrogen exchange can provide insight into both the population and the detailed structure of transient protein conformations. PMID:19722680

  1. Spectral and biological evaluation of a synthetic antimicrobial peptide derived from 1-aminocyclohexane carboxylic acid.

    PubMed

    Abercrombie, J J; Leung, Kai P; Chai, Hanbo; Hicks, Rickey P

    2015-03-15

    Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)KKKK-amide (A6c=1-aminocyclohexane carboxylic acid) is a synthetic antimicrobial peptide (AMP) that exhibits in vitro inhibitory activity against drug resistant strains of Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterococcus faecium at concentrations ranging from 10.9 to 43μM. Spectroscopic investigations were conducted to determine how this AMP interacts with simple membrane model systems in order to provide insight into possible mechanisms of action. CD and 2D-(1)H NMR experiments indicated this AMP on binding to SDS and DPC micelles adopts conformations with varying percentages of helical and random coil conformers. CD investigations in the presence of three phospholipid SUVs consisting of POPC, 4:1 POPC/POPG, and 60% POPE/21%POPG/19%POPC revealed: (1) The interactions occurring with POPC SUVs have minimal effect on the conformational diversity of the AMP yielding conformations similar to those observed in buffer. (2) The interactions with 4:1 POPC/POPG, and 60% POPE/21%POPG/19%POPC SUVs exhibited a greater influence on the percentage of different conformers contributing to the CD spectra. (3) The presence of a high of percentage of helical conformers was not observed in the presence of SUVs as was the case with micelles. This data indicates that the diversity of surface bound conformations adopted by this AMP are very different from the diversity of conformations adopted by this AMP on insertion into the lipid bilayer. CD spectra of this AMP in the presence of SUVs consisting of LPS isolated from P. aeruginosa, K. pneumoniae and Escherichia coli exhibited characteristics associated with various helical conformations.

  2. Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

    PubMed Central

    Sekirnik (née Measures), Angelina R.; Hewings, David S.; Theodoulou, Natalie H.; Jursins, Lukass; Lewendon, Katie R.; Jennings, Laura E.; Rooney, Timothy P. C.; Heightman, Tom D.

    2016-01-01

    Abstract A range of isoxazole‐containing amino acids was synthesized that displaced acetyl‐lysine‐containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4‐mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole‐containing peptides are comparable to those of a hyperacetylated histone H4‐mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole‐based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4‐mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl‐lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3. PMID:27264992

  3. Permeation of membranes by the neutral form of amino acids and peptides: relevance to the origin of peptide translocation

    NASA Technical Reports Server (NTRS)

    Chakrabarti, A. C.; Deamer, D. W.; Miller, S. L. (Principal Investigator)

    1994-01-01

    The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10(5) slower than facilitated inward transport across biological membranes. This suggest that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10(10) times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g. for certain signal sequences, toxins and thylakoid proteins) in vivo.

  4. Conjugates of amino acids and peptides with 5-o-mycaminosyltylonolide and their interaction with the ribosomal exit tunnel.

    PubMed

    Shishkina, Anna; Makarov, Gennady; Tereshchenkov, Andrey; Korshunova, Galina; Sumbatyan, Nataliya; Golovin, Andrey; Svetlov, Maxim; Bogdanov, Alexey

    2013-11-20

    During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.

  5. Amino Acid Chirality and Ferrocene Conformation Guided Self-Assembly and Gelation of Ferrocene-Peptide Conjugates.

    PubMed

    Adhikari, Bimalendu; Singh, Charanpreet; Shah, Afzal; Lough, Alan J; Kraatz, Heinz-Bernhard

    2015-08-03

    The self-assembly and gelation behavior of a series of mono- and disubstituted ferrocene (Fc)-peptide conjugates as a function of ferrocene conformation and amino acid chirality are described. The results reveal that ferrocene-peptide conjugates self-assemble into organogels by controlling the conformation of the central ferrocene core, through inter- versus intramolecular hydrogen bonding in the attached peptide chain(s). The chirality controlled assembling studies showed that two monosubstituted Fc conjugates FcCO-LFLFLA-OMe and FcCO-LFLFDA-OMe form gels with nanofibrillar network structures, whereas the other two diastereomers FcCO-DFLFLA-OMe and FcCO-LFDFLA-OMe exclusively produced straight nanorods and non-interconnected small fibers, respectively. This suggests the potential tuning of gelation behavior and nanoscale morphology by altering the chirality of constituted amino acids. The current study confirms the profound effect of diastereomerism and no influence of enantiomers on gelation. Correspondingly, the diastereomeric and enantiomeric Fc[CO-FFA-OMe]2 were constructed for the study of chirality-organized structures.

  6. Analgesic effects of mambalgin peptide inhibitors of acid-sensing ion channels in inflammatory and neuropathic pain.

    PubMed

    Diochot, Sylvie; Alloui, Abdelkrim; Rodrigues, Précillia; Dauvois, Mélodie; Friend, Valérie; Aissouni, Youssef; Eschalier, Alain; Lingueglia, Eric; Baron, Anne

    2016-03-01

    Mambalgins are 57-amino acid peptides isolated from snake venom that evoke naloxone-resistant analgesia after local (intraplantar) and central (intrathecal) injections through inhibition of particular subtypes of acid-sensing ion channels (ASICs). We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical inflammatory pain after systemic intravenous (i.v.) administration and are effective against neuropathic pain. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels, along with a contribution of ASIC1a-containing channels, in the i.v. effects of these peptides against inflammatory pain. The potent analgesic effect on neuropathic pain involves 2 different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection and an ASIC1a-dependent and partially naloxone-sensitive effect associated with intrathecal injection. These data further support the role of peripheral and central ASIC1-containing channels in pain, demonstrate their participation in neuropathic pain, and highlight differences in the repertoire of channels involved in different pain conditions. They also strengthen the therapeutic potential of mambalgin peptides that are active in a broader range of experimental pain models and through i.v. systemic delivery.

  7. Adsorption of Amino Acids and Peptides on Metal and Oxide Surfaces in Water Environment: A Synthetic and Prospective Review.

    PubMed

    Costa, D; Savio, L; Pradier, C-M

    2016-07-28

    Amino acids and peptides are often used as "model" segments of proteins for studying their behavior in various types of environments, and/or elaborating functional surfaces. Indeed, though the protein behavior is much more complex than that of their isolated segments, knowledge of the binding mode as well as of the chemical structure of peptides on metal or oxide surfaces is a significant step toward the control of materials in a biological environment. Such knowledge has considerably increased in the past few years, thanks to the combination of advanced characterization techniques and of modeling methods. Investigations of biomolecule-surface interactions in water/solvent environments are quite numerous, but only in a few cases is it possible to reach an understanding of the molecule-(water)-surface interaction with a level of detail comparable to that of the UHV studies. This contribution aims at reviewing the recent data describing the amino acid and peptide interaction with metal or oxide surfaces in the presence of water.

  8. Site-directed and global incorporation of orthogonal and isostructural noncanonical amino acids into the ribosomal lasso peptide capistruin.

    PubMed

    Al Toma, Rashed S; Kuthning, Anja; Exner, Matthias P; Denisiuk, Alexander; Ziegler, Juliane; Budisa, Nediljko; Süssmuth, Ro