Marker Gene Monthly Newsletter   

April, 2006

Volume 6, Number 4

© 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.

Gaussia Luciferase Assays.

Gaussia Luciferase is a new reporter luciferase isolated from the marine copepod Gaussia princeps. Gaussia luciferase can be expressed in mammalian cells using commercially available reporter plasmids.  This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine (M0739) in a reaction that emits light (at 470 nm), and has considerable advantages over other reporter genes.  Gaussia Luciferase possesses a natural secretory signal and upon expression is secreted into the cell medium of cells grown in culture. Therefore, cell lysis is not necessary for assaying expression levels.  In addition, the gene product has a molecular weight of only 19.9 kD (185 AA) making it one of the smallest known luciferases.  The enzyme also has a broad pH optimum at 7.7, with activity dependent upon the concentration of monovalent cations.  The enzyme is resistant to decomposition due to exposure to heat, cold, strongly acidic or basic conditions.  Analysis of the gene sequence indicates a secretory signal that functions in both prokaryotes and eukaryotes.  Most importantly, Gaussia luciferase generates over 1000-fold higher bioluminescent signal intensity, compared to Firefly and Renilla Luciferases, making it an ideal transcriptional reporter for cell culture assays.  The secreted protein is very stable and can be stored for several days at 4°C without significant loss of activity.   The substrate, coelenterazine is common to a number of marine bioluminescent reactions, including those from Renilla, Aequorea and Watesenia. In some of these reactions it is utilized as a simple substrate being catalytically turned over in the bioluminescent reaction, while in others, such as in the photoprotein systems of Mneiopsis, it is incorporated as part of the photoprotein.  The substrate h-coelenterate luciferin is a coelenterazine luciferin analog wherein one of the phenolic groups is replaced with a proton.  This substrate exhibits a high recharging efficiency and even higher light output.   Marker Gene is now providing these new coelenterazine substrates, as well as a convenient new assay kit MarkerGene^TM Gaussia Luciferase Cellular Assay Kit (M1193) for quickly measuring the luciferase activity in mammalian cell culture.  Please see our website or the references below for more information about these exciting new assays. 

•  McCapra and Beheshti in "Bioluminescence and Chemiluminescence: Instruments and Applications", 1985, Ed. K. Van Dyke, CRC Press, Boca Raton, FL. pgs. 9-42.
• Schuster, G.B., Schmidt, S.P. (1982) “Chemiluminescence of Organic Compounds” Adv. Phys. Org. Chem. 18: 187.
• Bakhos A. Tannous, Dong-Eog Kim, Juliet L. Fernandez, Ralph Weissleder and Xandra O. Breakefield (2005) “Codon-Optimized Gaussia Luciferase cDNA for Mammalian Gene Expression in Culture and in Vivo” Molecular Therapy, 11(3): 435-443.
• Verhaegent M, Christopoulos TK. (2002) “Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization.”  Anal. Chem. 74(17): 4378-85.
  

New Molecular Rotors.

The importance of membrane fluidity in cellular biology and physiology has led to the development of several fluorescence-based methods for its quantitative measurement in live cells.  Depending on the membrane viscosity (or membrane fluidity, which is its reciprocal), phospholipids and membrane-bound proteins can show different vertical, rotational and lateral diffusion behavior within the membrane.  Changes in membrane fluidity have been associated with alterations in cellular physiology, including membrane transport, activities of membrane-bound enzymes and receptor binding. In addition, variations in membrane viscosity have been linked to a variety of diseases, such as atherosclerosis, malignancy, hypercholesterolemia, diabetes, aberrant hepatic microcirculation, Alzheimer's disease and apoptosis.    Endothelial cells also appear to be able to sense fluid shear stress through the cell membrane and produce changes that help to maintain a specific level of blood flow within blood vessels.  Recently, Professor Emmanuel Theodorakis and collaborators at the Departments of Chemistry and Bioengineering at UCSD and Department of Biological Engineering at the University of Missouri have developed a series of new membrane viscosity probes based upon the fluorescent dye 9-(dicyanovinyl)-julolidine (DCVJ), that contain a long chain alkyl group through which they partition to the plasma membrane.  Exhibited fluorescence increases of up to 30-fold (viscosity-dependent fluorescence quantum yield) were found upon rotational changes in the probe environment (viscosity).   In addition, these researchers also developed several new ratiometric membrane fluidity probes based upon coupling with a second dye (i.e. 7-methoxycoumarin-3-carboxylic acid (MCCA), that could also act as a donor fluorophore via Resonance Energy Transfer (RET) techniques.  These new “molecular rotors” are expected to be useful in cellular high-throughput assay systems and for analysis of sheer stress and viscosity in cell membranes.  These new rotors will add to the arsenal of techniques already available (including our MarkerGene^TM Membrane Fluidity Kit, M0271) for analysis of these properties in cells grown in culture or in vivo.  For more information about these new methods, please see the references below visit our website.

• Haidekker, M.A., Ling, T., Anglo, M., Stevens, H.Y., Frangos, J. A., Theodorakis, E. A., (2001)  “New fluorescent probes for the measurement of cell membrane viscosity”, Chemistry & Biology 8: 123-131.
• Theodorakis, E. A., Haidekker, M.A., Brady, T., Wen, K., Okada, C., Stevens, H.Y., Snell, J.M., Frangos J.A., (2002) “Phospholipid-bound Molecular Rotors: Synthesis and Characterization.” Bioorg. & Med. Chem. 10: 3627-3636.
• Haidekker, M.A., Brady, T.P., Lichlyter, D., Theodorakis, E.A., (2005) “A Ratiometric Fluorescent Viscosity Sensor” J. Amer. Chem. Soc. 128: 398-399.

New Inhibitor of Lipase and b-Lactamase.

N-(5-dimethylaminonaphthalene-1-sulfonyl)-3-aminobenzeneboronic acid (M0329: Dansylaminophenylboronic acid) is a useful fluorescent derivative of m-aminophenylboronic acid that binds to cis-diols in carbohydrates or other molecules containing vicinal diols (e.g. geminal serines in proteins).  Interesting new properties for this compound have recently been revealed, as an active-site inhibitor of several important enzymes including lipoprotein lipase, human milk lipase, beta-lactamase, substilin, as well as other peptidases.  Resonance energy transfer has been used to identify the interaction of dansylaminophenylboronic acid with bile salt-stimulated human milk lipase (BSSL) and a binding constant of 8.6 X 10⁶ M-1 was measured.  Benzeneboronic acid competitively displaced dansylbenzeneboronic acid from the enzyme (Ki = 42 mM).  The same method was used to monitor the interaction of the active-site-directed fluorescent inhibitor with lipoprotein lipase (EC 3.1.1.34). The binding to the active site of lipoprotein lipase had an association constant, Ka, of 1.1 X 10⁶ M-1, indicating a strong interaction. The binding was also displaced competitively by benzene boronic acid.

Interestingly, this same compound (dansylbenzeneboronic acid, M0329) has recently also been found to inhibit AmpC b-lactamase activity.  The expression of b-lactamases is the most common form of bacterial resistance to b-lactam antibiotics. To combat these enzymes, agents that inhibit (e.g. clavulanic acid) or evade (e.g. aztreonam) b-lactamases have been developed.  However, both the b-lactamase inhibitors and the b-lactamase-resistant antibiotics are themselves b-lactams, and bacteria have responded to these compounds by expressing variant enzymes resistant to inhibition (e.g. IRT-3) or that inactivate the b-lactamase-resistant antibiotic (e.g. TEM-10).  Moreover, these compounds also serve to select for bacteria that have intrinsically resistant b-lactamases (e.g. AmpC).   In an effort to identify non-b-lactam-based b-lactamase inhibitors, crystallographic structural analysis was  used to identify several new m-aminophenylboronic acid b-lactamase complexes as potential inhibitors of this enzyme.  These data suggested that modifications of the base structure might enhance the affinity of boronic acid-based inhibitors for class C b-lactamases (e.g. Escherichia coli AmpC).  Among the most potent inhibitors identified were dansylaminophenylboronic acid (Ki = 1.3 mM), with the most potent of these compounds, benzo[b]thiophene-2-boronic acid having an affinity for E. coli AmpC of 27 nM.   In addition, preliminary evidence suggests that these new boronic acid-based inhibitors can potentiate the activity of current b-lactam antibiotics, such as amoxicillin and ceftazidime, against bacteria expressing class C b-lactamases. This data suggests that boronic acid-based compounds may serve as leads for the development of new therapeutic agents for the treatment of b-lactam-resistant infections.  For more information about these new inhibitors, please see the references below, or visit our website.

• O'Connor, C. J.; Yaghi, B. M. (1989) “N-(5-dimethylaminonaphthalene-1-sulfonyl)-3-aminobenzene boronic acid as an active-site-directed fluorescent probe of bile-salt-stimulated human milk lipase.” Journal of Molecular Catalysis 52(3): 317-321.
• Vainio P. (1983)   “N-(5-dimethylaminonaphthalene-1-sulfonyl)-3-aminobenzene boronic acid as an active-site-directed fluorescent probe of lipoprotein lipase.” Biochimica et biophysica acta 746(3): 217-219.
• Weston, G. S., Blazquez, J.,  Baquero, F.,  Shoichet, B. K. (1998), “Structure-Based Enhancement of Boronic Acid-Based Inhibitors of AmpC b -Lactamase.” Journal of Medicinal Chemistry 41(23): 4577-4586.
• Shoichet, B. K.; Costi, M.P., Tondi, D., (2000) “Phenylboronic acid derivative inhibitors of b -lactamases, their preparation, pharmaceutical compositions, and therapeutic use.” PCT Int. Appl. (2000), 41 pp.
• Philipp M., Maripuri S., (1981), “Inhibition of subtilisin by substituted arylboronic acids.” FEBS letters 133(1): 36-8.

Microsomal Dealkylase and Cytochrome P450 Measurement in Live Cells.

The cytochome P-450’s are a large group of monooxygenase enzymes responsible for the metabolism of toxins in the body.  These enzymes, requiring NADPH as a cofactor and O₂ as co-substrate, are located in the endoplasmic reticulum and are highly concentrated in the liver and small intestine.  Additionally, Cytochrome P-450’s are also found in the mitochondrial membrane.  P-450 enzymes encompass a highly diverse "superfamily" of hemoproteins, and metabolic oxidation of chemical compounds is the main function of the cytochrome-mediated monooxygenase or mixed-function oxidase system.  Cytochrome P450 is also used as a marker for the endoplasmic reticulum.  The active site of cytochrome P450 contains the catalyst heme (iron-porphyrin) with a sulfur of cysteine serving as the fifth iron ligand.  Although these enzymes typically have a low turnover rate, their intracellular activity can be monitored using fluorogenic alkyl ether derivatives of fluorophores including resorufin.  Marker Gene has now introduced the resorufin ether substrate 7-methoxyresorufin (M1166) that is one of the most popular substrates for analysis of microsomal dealkylase and cytochrome P450 enzymes.  It produces the red fluorescent dye resorufin (EX/EM 571/585 nm) upon enzyme activity.  This substrate has also been extensively used to differentiate the various isozymes of the Cytochrome P450 system.  For more information about these assays, please see the references below or visit our website.

• Sugihara K., Kitamura S., Yamada T., Okayama T., Ohta S., Yamashita K., Yasuda M., Fujii-Kuriyama Y., Saeki K., Matsui S., Matsuda T., (2004) “Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo.” Biochem Biophys Res Commun.;318(2): 571-578.
• Haasch M.L., Graf W.K., Quardokus E.M., Mayer R.T., Lech J.J  (1994) "Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers." Biochem Pharmacol 47: 893-903.
• Nerurkar P.V., Park S.S. Thomas P.E., Nims R.W., Lubet R.A., (1993) "Methoxyresorufin and benzyloxyresorufin: substrates preferentially metabolized by cytochromes P4501A2 and 2B, respectively, in the rat and mouse." Biochem. Pharmacol. 46: 933-943.
• Wolf C.R., Seilman S., Oesch F., Mayer R.T., Burke M.D., (1986) "Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins." Biochem. J. 240:  27-33.
  
  

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.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you.