Feb

23

Peptide Arrays – Protocol

Filed Under Application Note | Leave a Comment

11. Antimicrobial Peptide Arrays for Detection of Inactivated Biothreat Agents By: Chris R. Taitt1 , Stella H. North1, Nadezhda V. Kulagina1

Arrays of immobilized antimicrobial peptides are used to detect bacterial, viral, and rickettsial pathogens, including inactivated biothreat agents. These arrays differ from the many combinatorial peptide arrays described in the literature in that the peptides used here have naturally evolved to interact with and disrupt microbial membranes with high affinity but broad specificity. The interaction of these naturally occurring peptides with membranes of pathogens has been harnessed for the purpose of detection, with immobilized antimicrobial peptides acting as “capture” molecules in detection assays. Methods are presented for immobilizing the antimicrobial peptides in planar arrays, performing direct and sandwich assays, and detecting bound targets.

Affiliation(s): (1) US Naval Research Laboratory, Washington, DC, USA

Book Title: Peptide Microarrays: Methods and Protocols Series: Methods in Molecular Biology | Volume: 570 | Pub. Date: Aug-01-2009 | Page Range: 233-255 | DOI: 10.1007/978-1-60327-394-7_11

Subject: Protein Science

Key Words: Biothreat – detection, array – antimicrobial peptide

Feb

15

New Customer Publication – MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

Filed Under New Customer Publications | Leave a Comment

MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20–22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm2, 12 h) regulated the EC expression of manymiRs, including miR-19a.We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3′-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

Qin X, Wang X, Wang Y, Tang Z, Cui Q, Xi J, J Li YS, Chien S, Wang N. (2010) MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells. Proc Natl Acad Sci USA [Epub ahead of print]. [abstract]

Feb

12

Peptide Microarray Publication – Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds

Filed Under New Customer Publications | Leave a Comment

Williams BA, Diehnelt CW, Belcher P, Greving M, Woodbury NW, Johnston SA, Chaput JC. (2009) Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds. J Am Chem Soc 131(47), 17233-41. [abstract]

A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4000 unique 12-mer peptides was screened to identify sequences that bind to nonoverlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of  ~1000-fold (ΔΔG = ~4.1 kcal/mol) between the individual peptides and final bivalent synbody construct.

Feb

10

The many complex roles of microRNA in breast cancer

Filed Under Technical Article | 1 Comment

There have been several studies involving microRNAs and breast cancer to date.  The findings of these studies collectively demonstrate the wide range of function that microRNA can play in just a single disease.  Researchers at Baylor College of Medicine and the University of Houston found that dysregulation of certain microRNAs have significant effect on morphological and molecular changes such as an expansion of the progenitor cell population, decreased cell size, increased cellular proliferation, and colony-forming potential [1]. They suggest that the dysregulation of these microRNAs might be important in the causation, or origination of breast cancer.

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer.  Researchers at the University of Lethbridge, Canada found that chemoresistance may be linked to drug-induced dysregulation of microRNA function [2]. They found many dysregulated microRNAs in drug resistant MFC-7 human breast adenocarcinoma cells and that some of these microRNAs target a human multidrug resistance-associated protein. Their results suggest that dysregulated microRNA expression may underlie the abnormal functioning of critical cellular processes associated with the chemoresistance phenotype.

A major complication of breast cancer is its metastatic potential and now, some new studies have shown that microRNAs can affect breast cancer metastasis either by over or under expression.  In this first example, researchers show that over expression of a specific microRNA plays a key role in a cancer signaling pathway. It is known that Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility and that aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Researchers at Tufts University found that addition of an anti-miR can block the ROCK signaling pathway resulting in decreased breast cancer cell invasion/ migration and metastasis [3].

In another study, it was found that microRNAs function in an opposite manner. Researchers at Memorial Sloan-Kettering Cancer Center found a specific set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential [8]. Furthermore, they show that restoring the expression of these microRNAs in malignant cells reduces overall tumor growth and proliferation and suppresses metastatic cell invasion.

  1. Greene SB, Gunaratne PH, Hammond SM, Rosen JM. (2010) A putative role for microRNA-205 in mammary epithelial cell progenitors. J Cell Sci [Epub ahead of print] [abstract]
  2. Pogribny IP, Filkowski JN, Tryndyak VP, Golubov A, Shpyleva SI, Kovalchuk O. (2010) Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin. Int J Cancer [Epub ahead of print] [abstract]
  3. Liu S, Goldstein RH, Scepansky EM, Rosenblatt M.  (2009) Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone.  Cancer Res 69(22), 8742-751.   [abstract]
  4. Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J.  (2009) miR-200 enhances mouse breast cancer cell colonization to form distant metastases.  PLoS One 4(9), e7181.  [abstract]
  5. Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM. (2009) Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids 37(8), 2584-95.  [abstract]
  6. Sun Y, Wu J, Wu SH, Thakur A, Bollig A, Huang Y, Joshua Liao D. (2008) Expression profile of microRNAs in c-Myc induced mouse mammary tumors. Breast Cancer Res Treat 118(1), 185-96.  [abstract]
  7. Kovalchuk O, Filkowski J, Meservy J, Ilnytskyy Y, Tryndyak VP, Chekhun VF, Pogribny IP. (2008) Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin. Mol Cancer Ther 7(7), 2152-59.  [abstract]
  8. Tavazoie SF, Alarcón C, Oskarsson T, Padua D, Wang Q, Bos PD, Gerald WL, Massagué J. (2008) Endogenous human microRNAs that suppress breast cancer metastasis. Nature 451(7175), 147-52.  [abstract]
  9. Kovalchuk O, Tryndyak VP, Montgomery B, Boyko A, Kutanzi K, Zemp F, Warbritton AR, Latendresse JR, Kovalchuk I, Beland FA, Pogribny IP. (2007) Estrogen-induced rat breast carcinogenesis is characterized by alterations in DNA methylation, histone modifications and aberrant microRNA expression. Cell Cycle 6(16), 2010-18.  [abstract]

Feb

3

Recent Customer Publications – microRNA

Filed Under New Customer Publications | Leave a Comment

  1. Greene SB, Gunaratne PH, Hammond SM, Rosen JM. (2010) A putative role for microRNA-205 in mammary epithelial cell progenitors. J Cell Sci [Epub ahead of print] [abstract]
  2. Pogribny IP, Filkowski JN, Tryndyak VP, Golubov A, Shpyleva SI, Kovalchuk O. (2010) Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin. Int J Cancer [Epub ahead of print] [abstract]
  3. Sluijter JP, van Mil A, van Vliet P, Metz CH, Liu J, Doevendans PA, Goumans MJ. (2009) MicroRNA-1 and -499 Regulate Differentiation and Proliferation in Human-Derived Cardiomyocyte Progenitor Cells. Arterioscler Thromb Vasc Biol [Epub ahead of print] [abstract]
  4. Curtale G, Citarella F, Carissimi C, Goldoni M, Carucci N, Fulci V, Franceschini D, Meloni F, Barnaba V, Macino G. (2009) An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes. Blood [Epub ahead of print] [abstract]
  5. Filkowski J, Ilnytskyy Y, Tamminga J, Koturbash I, Golubov A, Bagnyukova T, Pogribny I, Kovalchuk O. (2009) Hypomethylation and genome instability in the germline of exposed parents and their progeny is associated with altered miRNA expression. Carcinogenesis [Epub ahead of print] [abstract]
  6. Liu W, Gong YH, Chao TF, Peng XZ, Yuan JG, Ma ZY, Jia G, Zhao JZ. (2009) Identification of differentially expressed microRNAs by microarray: a possible role for microRNAs gene in medulloblastomas. Chin Med J (Engl) 122(20), 2405-411. [abstract]
  7. Duan Z, Choy E, Nielsen GP, Rosenberg A, Iafrate J, Yang C, Schwab J, Mankin H, Xavier R, Hornicek FJ. (2009) Differential expression of microRNA (miRNA) in chordoma reveals a role for miRNA-1 in Met expression. J Orthop Res [Epub ahead of print] [abstract]
  8. Lau P, Verrier JD, Nielsen JA, Johnson KR, Notterpek L, Hudson LD. (2008) Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes. J Neurosci 28(45), 11720-730. [abstract]