Marker Gene Monthly Newsletter   

January, 2006

Volume 6, Number 1

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Pigmentation Gene Localized in MNT1 Cells.

Skin coloration is a result of a complex process involving specialized pigment cells and production of the insoluble polymeric protein melanin in melanosomes.  A gene (slc24a5) that appears to be responsible for coloration changes in this process has been isolated from zebrafish (phenotype golden) by an international team headed by Dr. Keith Cheng at the Penn State University College of Medicine and found to be mutant in this lightly colored variety.  Since this gene shares extensive homology with the human gene, the researchers also found that they were able to restore normal “dark” pigmentation in the golden zebrafish by injecting human mRNA for slc24a5 into golden embryos.  slc24a5 codes for a calcium ion transport protein that affects intracellular organelle calcium ion concentrations.  The identity and localization of this gene and it’s protein product(s) were defined using fusion proteins with the marker gene GFP or an HA (hemagglutinin) marker (A triple HA-tag fusion at the C-terminus of zebrafish slc24a5).   The HA tag was visualized using a monoclonal anti-HA antibody and a Cy3-conjugated secondary goat anti-mouse antibody.   These studies indicated that the gene products were localized in subcellular compartments in MNT1 cells (a constitutively pigmented human melanoma cell line).  The results of these studies shed light on some of the important processes of pigmentation in mammalian cells.  For more information about these methods and systems for identifying gene products in vivo, please see our website or consult the references below. 

•   Canfield, V. A., Levenson, R., (1993) “Transmembrane organization of the Na,K-ATPase determined by epitope addition.” Biochemistry, 32: 13782-13786 (1993).
• Lamason, R.L., Mohideen, M-A, P.K., Mest, J.R.,  Wong, A.C., Norton, H.L., Aros, M.C., Jurynec, M.J., Mao, X., Humphreville, V.R., Humbert, J.E., Sinha, S., Moore, J.L., Jagadeeswaran, P.,  Zhao, W., Ning, G., Makalowska, I., McKeigue, P.M., O’Donnell, D., Kittles, R., Parra, E.J., Mangini, N.J., Grunwald, D.J., Shriver, M.D., Canfield, V.A., Cheng K.C., “SLC24A5, a Putative Cation Exchanger, Affects Pigmentation in Zebrafish and Humans” Science 310(5755): 1782 – 1786.
• Smith D.R., Spaulding D.T., Glenn H.M., Fuller B.B.,  (2004) “The relationship between Na(+)/H(+) exchanger expression and tyrosinase activity in human melanocytes.” Exp. Cell Res. 298(2): 521-34.
  

FRET for Monitoring of Peptide and Protein Interactions.

Fluorescence energy transfer (FRET) between a donor and an acceptor molecule is a powerful technique that has been widely applied in the investigation of interactions between or even within peptides and proteins.  In this method, the fluorescence from one fluorophore either excites another nearby fluorophore, or it’s fluorescence is blocked by a quencher that is bound to a nearby site on the protein or peptide.  Alternately, two proteins can be labeled and their binding monitored through their specific receptor-ligand binding.  In addition, changes in protein structure or enzyme activity can be determined by monitoring these proximity interactions.  Methods include internal quenching and changes in intensity measurements, donor or acceptor reduction kinetics, fluorescence lifetime or emission anisotropy measurements.  The combination of these techniques have made for very powerful analysis methods for use in biology and biochemistry.  Because the degree of interaction in FRET decreases quickly with distance, only interactions of close proximity are typically measured. 

One of the most common FRET methods involves the design of enzyme substrates based upon quenching of fluorescence by attaching a matched quencher-fluorophore pair to adjacent sites on a peptide or protein.  Upon enzymatic cleavage, these labels become separated.  These type of substrates are especially useful for measurement of endopeptidase activities.   Labeled peptides can be prepared by modifying existing peptides or even by incorporating the labels during solid-phase peptide synthesis.  Strategies used to label peptides during synthesis require dyes or quenchers that are not damaged by deblocking procedures or covalent modification on specific residues following synthesis.   For example, synthetic peptides may be covalently labeled by amine- or thiol-reactive protein labels.  Fluorophores or quenchers can even be conjugated to the N-terminus of a resin-bound peptide before other protecting groups are removed and the labeled peptide is released from the resin.  Marker Gene provides a variety of reactive fluorescein (M0955), tetramethylrhodamine (M0972), EDANS (M0273), coumarin (M1053), eosin (M1054) or biotin labels (M0783, M0785) that are stable enough to resist the chemical deprotection conditions.  In addition, our Dabcyl and Mancyl  quenchers (M1051 or M0532) are well suited for use in preparing quenched substrates as FRET pairs.  Another possibility is the use of biotin labeling, which allows secondary specific binding by streptavidin or avidin-conjugates to the labeled site.  For more information about these exciting possibilities for measuring protein or peptide interactions using FRET, please see our website or see the references below

• Marmé, N., Knemeyer J. P., Sauer, M., Wolfrun, J. (2003) “Inter- and Intramolecular Fluorescence Quenching of Organic Dyes. Bioconjugate Chem. 14: 1133.
• Marmé, N., Knemeyer, J. P., Wolfrun, J., Sauer, M., (2004) “Highly Sensitive Protease Assay Using Fluorescence quenching of Peptide Probes Based on Photoinduced Electron Transfer.” Angew. Chem., Int. Ed. Engl. 43: 3798.
• Kenworthy, A. K. (2001) “Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.” Methods 24: 289.
• Hoppe, A., Christensen, K., Swanson, J. A., (2002) “Imaging protein-protein interactions in living cells.” Biophys. J. 83: 3652.
• Ozawa, T., Umezawa, Y., (2002) “Peptide assemblies in living cells. Methods for detecting protein-protein interactions.” Supramol. Chem. 14: 271.
• Wieb van der Meer, B., Coker, G., Simon, C. (1994) “Resonance Energy Tranfer: Theory and Data.” VCH: New York.
• Young, R. M., Arnette, J. K., Roess, D. A., Barisas, B. G. (1994) “Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.” Biophys. J. 67: 881.
• Chicester, U. K., Andrews D. L., Demidov A. A., (1999) “Resonance Energy Transfer.” Wiley & Sons: New York, 1999.
• Widengren, J., Schweinberger, E., Berger, S., Seidel, C. A. M. (2001) “Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: theory and experimental realizations.” J. Phys. Chem. A 105: 6851.
• Clayton, A. H. A., Hanley, Q. X. Arndt-Jofin D. J., Subramaniam, V., Jovin, T. M. (2002) “Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM) Biophys. J. 83: 1631.
• Clegg, R. M, Holub, O., Gohlke, C. (2003) “Fluorescence lifetime-resolved imaging: measuring lifetimes in an image.” Methods Enzymol. 360: 509.
• Jares-Erijman, E. A., Jovin, T. M. (2003) FRET imaging. Nat. Biotechnol. 21: 1387–1395.
• within phospholipid membranes.” Biochemistry 33: 7211.
• Ben-Efraim, I., Strahilevitz, J., Bach, D., Shai, Y., (1994) “Secondary structure and membrane localization of synthetic segments and a truncated form of the IsK (minK) protein.” Biochemistry 33:  6966.
• Matayoshi, E. D., Wang, G. T., Krafft G. A., Erickson, J. (1990) “Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer.” Science 247: 954.
• Wang, G. T. (1990) “Design and Synthesis of New Fluorogenic HIV Protease Substrates Based on Resonance Energy Transfer.” Tetrahedron Lett. 31: 6493.
• Garcia-Echeverria, C., Rich, D. H. (1992) “New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.” FEBS Lett. 297: 100.
• Wang, G. T., Krafft, G. A. (1992) “Automated Synthesis of Fluorogenic Protease Substrates: Design of Probes for Alzheimers Disease-Associated Proteases.” Bioorg. Med. Chem. Lett. 2: 1665.
• Maggiora, L. L., Smith, C. W., Zhang, Z. Y. (1992) “A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis.”  J. Med. Chem. 35: 3727.
• Contillo, L. G., et al. General Strategy for the Synthesis of Eosin Fluorescein Energy Transfer Substrates for High Sensitivity Screening of Protease Inhibitors. In “Techniques in Protein Chemistry V”, Crabb J. W., Ed. Academic Press: New York, 1994, pp 493.
• Weder, J. K. P, Kaiser, K-P. (1995) “Fluorogenic Substrates for Hydrolase Detection Following Electrophoresis.” J. Chromatogr. A 698: 181.

ROS measurement in mitochondria.

The mitochondria plays a central role in cell function, producing over 95% of cellular ATP requirements, regulating various cellular processes and physiology and it has been implicated in numerous disease states including neurodegenerative disorders, carcinogenesis, injuries from ischemic reperfusion, atherosclerosis as well induction from xenobiotic toxicity events.  Cellular reactive oxygen species (ROS) production has proved to be a useful parameter of mitochondrial function as increased ROS activity has been implicated in various disease states.  The degree of ROS production can be assessed by loading mitochondria with reduced dyes that, upon reaction with these ROS, produce highly fluorescent derivatives. 

Dichlorofluorescin, diacetate (H₂DCFDA, M0807) is such a cell-permeant indicator for reactive oxygen species that is nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell.  The reduced substrate releases the highly fluorescent dye 2',7'-dichlorofluorescein and allows easy detection inside living cells.  When the cell begins to produce reactive oxygen species, the highly fluorescent dye 2',7'-dichlorofluorescein is produced, with EX: 495nm and EM: 529 nm.  Marker Gene now provides this reagent in an easy to use kit form in the MarkerGene^TM Live Cell Fluorescent Reactive Oxygen Species Detection Kit (M1049), that includes an inducer (for positive control applications), the H₂DCFDA substrate, standards and a detailed protocol for quick and easy discrimination of oxidatively stressed and nonstressed cells by fluorescence microscopy, microtiterplate assay or photomicroscopy.  For more information about this new kit, please see the references below or visit our website:

• Ubezio P., Civoli F., (1994) "Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells." Free Radic Biol Med 16: 509-516.
• Bailey S.R., Mitra S., Flavahan S., Flavahan N.A., (2005) "Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries." Am J Physiol Heart Circ Physiol 289: H243-50.
• Halliwell B., Whiteman M., (2004) "Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?" Br J Pharmacol 142: 231-55.
• Kutuk O., Adli M., Poli G., Basaga H., (2004) "Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts." Biofactors 20: 1-10.
• Myhre O., Andersen J.M., Aarnes H., Fonnum F., (2003) "Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation." Biochem Pharmacol 65: 1575-82.

New Proline Derivative for Use in Peptidomimetic Studies.

Proline is a non-polar amino acid that forms a tertiary amide bond when incorporated into peptides.  Since it does not have a hydrogen on the amide group, it cannot act as a hydrogen bond donor in protein structures.  This restricts the conformational space of the peptide chain in protein and peptide structures and tends to inhibit both α-helical and β-sheet structure formation.  Nevertheless, proline has been found in the transmembrane domains of many protein transporters and channels, regions believed to be α-helical in nature.  Many synthetic proline analogues have been developed that provide further restrictions of the proline amide bond conformation. Such proline mimetics are usually based on ring incorporation of heteroatoms into the ring, or the expansion or contraction of the proline ring.  These analogues are promising candidates for conformational studies and for tuning the biological, pharmacological, or physicochemical properties of naturally occurring proteins or peptides, as well as in the design of linear or cyclic peptide structures.  Several proline analogs and homologs are naturally occurring.  For example, trans-3-hydroxyproline and trans-4-hydroxyproline are constituents of collagens as a result of post-translational hydroxylation.  A new proline analog, trans-4-amino-L-Proline, has been developed that has shown interesting properties as a component of integrin binding antagonists, in hepatitis C viral inhibition, in CB1 receptor binding inhibition and for the preparation of novel enzyme inhibitors.  Marker Gene now provides a critical synthetic intermediate, N-Boc-trans-4-N-Fmoc-amino- L -proline (M1019), that can be used to incorporate the 4-amino-L-Proline molecule into numerous peptide synthesis schemes. For more information about these exciting new peptidomimetics, or the new products available from Marker Gene, please visit our website or see the reference below.

• Pepinsky, R. B., Mumford, R. A.; Chen, L. L., Leone, D., Amo, S. E., Van Riper, G., Whitty, A., Dolinski, B., Lobb, R. R., Dean, D. C., Chang, L. L., Raab, C. E., Si, Q., Hagmann, W. K., Lingham, R. B.,  (2002) “Comparative assessment of the ligand and metal ion binding properties of integrins a9b1 and a4b1.”    Biochemistry  41(22): 7125-7141
• Wang, Xiangdong Alan; Sun, Li-Quang; Sit, Sing-Yuen; Sin, Ny; Scola, Paul Michael; Hewawasam, Piyasena; Good, Andrew Charles; Chen, Yan; Campbell, Jeffrey Allen.  “Preparation of peptides as hepatitis C virus inhibitors.”    PCT Int. Appl.  (2003), 675 pp.  WO  2003099274 
• Moritani, Yasunori; Furukubo, Shigeru; Tsuboi, Yasunori; Okagaki, Chieko; Oku, Akira; Hirano, Naomitsu.  “Preparation of pyrrolidine derivatives as CB1 receptor antagonists.”   PCT Int. Appl.  (2005), 205 pp.  WO  2005115977
• Leftheris, Katerina.  “Preparation of amino acid and peptide amides as inhibitors of farnesyl protein transferase.”    Eur. Pat. Appl.  (1996), 73 pp.  EP  696593
• Saksena, Anil K.; Girijavallabhan, Viyyoor Moopil; Lovey, Raymond G.; Jao, Edwin E.; Bennett, Frank; McCormick, Jinping; Wang, Haiyan; Pike, Russell E.; Bogen, Stephane L.; Liu, Yi-Tsung; Arasappan, Ashok; Parekh, Tejal; Pinto, Patrick A.; Njoroge, F. George; Ganguly, Ashit K.; Brunck, Terence K.; Kemp, Scott Jeffrey; Levy, Odile Esther; Lim-Wilby, Marguerita.  Preparation of novel peptides as NS3-serine protease inhibitors of hepatitis C virus.    PCT Int. Appl.  (2002), 197 pp.  WO  2002008256.
• Olsen, B. R., Ninomiya, Y. In “Guidebook to the Extracellular Matrix and Adhesion Proteins”, Kreis, T., Vale, R., Eds, Oxford University Press: Oxford, 1993, p 40.
• Mauger, A.B., In “Chemistry and Biochemistry of Amino Acids, Peptide and Proteins”, Weinstein, B., Ed., Marcel Dekker: New York, 1977, p 179.
  
  

Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

Marker Gene Accepts Major Credit Cards.

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