Resorufin-Mannoside for Analysis of Golgi a-Mannosidase Activity.

The N-glycosylation pathway is a target for chemical intervention in a number of pathological conditions including cancer. Cells that have undergone oncogenic transformation often display abnormal cell surface oligosaccharides, and these changes in glycosylation are important determinants of tumor progression. Inhibition of the mannose trimming enzyme Golgi a-mannosidase II (GMII; mannosyl-oligosaccharide 1,3-1,6-a-mannosidase II; E.C. 3.2.1.114), the final glycoside hydrolase in the N-glycosylation pathway responsible for the formation of the “core” tri-mannose structure, has become a potential target for blocking these changes in cell surface oligosaccharides. Golgi a-mannosidase II (GMII) is thus a target for a number of synthetic efforts aimed at providing more selective and effective inhibitors.  Initial clinical tests indicated that the GMII inhibitor swainsonine (which inhibits in the nanomolar range) had promise as a cancer treatment agent.  However, side effects, possibly caused by the inhibition of Lysosomal Mannosidases by swainsonine, severely limited its usefulness, necessitating the search for more specific inhibitors.

The pH optimum of GMII is low (pH 5.7), and existing fluorogenic substrates like 4-methylumbelliferyl α-D-mannopyranoside (4MU-Man) cannot be used for continuous fluorometric measurement. The high pKa value of the released product, 4-methylumbelliferone (pKa of 7.8) makes it not substantially fluorescent at the low pH values found in the ER and Golgi. Chromogenic α-Mannosidase substrates like p-nitrophenyl α-D-mannopyranoside (PNP-Man) have also been used to monitor activity but they suffer from low sensitivity, and the absorbance readings of released pNP also cannot be recorded continuously at low pH, since pNP has a pKa of 7.1. And 5-bromo-4-chloro-3-indolyl α-D-mannopyranoside (X-Man) is a chromogenic substrate that releases a precipitating blue dye upon cleavage, and while activity can be observed at low pH, the solid nature of the product makes activity measurements very difficult to quantitate.  In order to overcome these limitations, Marker Gene has introduced a long wavelength fluorescent substrate, resorufin α-D-mannopyranoside (Res-Man, M1340), that can be utilized to obtain continuous fluorometric measurement of α-mannosidase activity.  This substrate releases the red fluorescent fluorophore Resorufin (EX:571; EM:585) with pKa  of 5.8.  Use of resorufin as the released product for low pH enzymes has previously been found effective in measuring cellulase activity continuously.  We believe this new Res-Man (M1340) substrate can be used to provide continuous fluorometric measurement, kinetic analysis, and inhibition screening of α-mannosidase activity at physiological pH values. For more information about this new substrate or allied assays, please see the references below, or visit our website.  

• Coleman DJ, Studler MJ, Naleway JJ (2007) "A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity:  resorufin-ß-D-cellobioside.", Anal. Biochem. 371:146-153.
• Rabouille C, Kuntz DA, Lockyer A, Watson R, Signorelli T, Rose DR, van den Heuvel M, Roberts DB (1999) "The Drosophila GMII gene encodes a Golgi a-mannosidase II.", J. Cell Sci. 112: 3319-3330.    
• Fiaux H, Kuntz DA, Hoffman D, Janzer RC, Gerber-Lemaire S, Rose DR, Juillerat-Jeanneret L (2008) "Functionalized pyrrolidine inhibitors of human type II a-mannosidases as anti-cancer agents:  Optimizing the fit to the active site.", Bioorganic and Medicinal Chemistry 16: 7337-7346.
• Vázquez-Reyna AB, Bálcazar-Orozco R, Flores-Carreón A (1993) "Biosynthesis of glycoproteins in Candida albicans:  Biochemical characterization of a soluble a-mannosidase.", FEMS Microbio. Lett. 106: 321-326.
• Bernacki RJ, Niedbala MJ, Korytnyk W, (1985) "Glycosidases in cancer and invasion." Cancer Meta. Rev. 4: 81-101.
• Dennis JW, Granovsky M, Warren CE, (1999) "Glycoprotein glycosylation and cancer progression." Biochim. Biophys. Acta 1473: 21-34.
• Hakomori S, (2002) "Glycosylation defining cancer malignancy: new wine in an old bottle." Proc. Nat. Acad. Sci. USA 99:10231-10233.
• Dube DH, Bertozzi CR (2005) "Glycans in cancer and inflammation. Potential for therapeutics and diagnostics." Nat. Rev. Drug Disc. 4: 477-488.
• Goss PE, Baker ME, Carver JP, Dennis JW (1995) "Inhibitors of carbohydrate processing: A new class of anticancer agents." Clin. Cancer Res. 1: 935-944.
• Moremen KW, Touster O, Robbins PW (1991) "Novel purification of the catalytic domain of Golgi a-mannosidase II. Characterization and comparison with the intact enzyme." J. Biol. Chem. 66: 16876-16885.
• van den Elsen JM, Kuntz DA, Rose DR (2001) "Structure of Golgi alpha-mannosidase II: a target for inhibition of growth and metastasis of cancer cells." EMBO J. 20: 3008-3017
• Zhong W, Kuntz DA, Ember B, Singh H, Moremen KW, Rose DR,  Boons GJ, (2008) "Probing the substrate specificity of Golgi a-mannosidase II using synthetic oligosaccharides and a catalytic nucleophile mutant." J. Am. Chem. Soc. 13: 8975-8983.
• Shah N, Kuntz DA, Rose DR, (2008) "Golgi a-mannosidase II cleaves two sugars sequentially in the same catalytic site." Proc. Natl. Acad. Sci. USA. 105: 9570-9575.
• Coleman DJ, Kuntz DA, Venkatesan M,  Cook GM, Williamson SP, Rose DR, Naleway JJ, (2009) "A Long Wavelength Fluorescent Substrate for Continuous Fluorometric Determination of α-Mannosidase Activity: Resorufin α-D-Mannopyranoside." Anal. Biochem. (in press).

Lentiviral Transfection in Mammalian Tissues.

Lentiviral transfection systems have gained steady acceptance for use in potential gene therapy applications since they have several key advantages over other transfection mechanisms. First they can be used to target cell transfection directed by the glycoprotein specificity of their envelope proteins. In addition, they permanently integrate into the host cell genome, and upon integration express their encoded viral proteins at high titer. Another important characteristic is their ability to transduce nondividing cells, as well as dividing cells in contrast to other retroviruses that transduce only dividing cells. Lentiviral vectors also hold two key advantages over AAV vectors. First, lentiviruses allow for a larger packaging capacity (8–10 kb) compared to less than AAV (~5Kb) and secondly, the majority of self-inactivating (SIN) HIV-based infective lentiviral particles become integrated into the genome within 3 days after infection compared to less than 10% for AAV. Because of these advantages, lentiviruses appear ideal for studies aimed at manipulating gene expression in mammalian cells and tissues.

Recent work from the laboratory of Dr. Greg Dissen and co-workers at the Division of Neuroscience, Oregon National Primate Research Center-Oregon Health & Science University have developed an HIV-1 based lentiviral system for gene therapy and other applications in mammalian systems. This system consists of a replication-incompetent, non-pathogenic version of the virus where the cis- and trans-acting components required to generate an infective viral particle are separated onto different plasmids. This results in virus particles that, upon infection, cannot propagate further infection. The vector cassette is a stripped version of the original HIV genome containing less than 5% of the parental genome. The marker gene eGFP is often co-cloned in these cassettes in order to track and quantitate transfection efficiency. For propagation, the packaging cell line 293T/17 (ATCC#CRL-11268), a highly transfectable derivative of the 293 human fetal kidney cell line, is used. For more information about these new and efficient lentiviral transfection systems, please visit our website or see the references below:

• Dissen GA, Lomniczi A, Neff TL, Hobbs TR, Kohama SG, Kroenke CD, Galimi F, Ojeda SR, (2009) "In vivo manipulation of gene expression in non-human primates using lentiviral vectors as delivery vehicles." Methods 49(1): 70-77
• Verma IM, Somia N, (1997) "Gene therapy - Promises, problems and prospects." Nature 389:239–242.
• Kafri T, (2004) "Gene delivery by lentivirus vectors an overview." Methods Mol. Biol. 246: 367–390.
• Thomas CE, Ehrhardt A, Kay MA, (2003) "Progress and problems with the use of viral vectors for gene therapy." Nat. Rev. Genet. 4: 346–358.
• Wong LF, Goodhead L, Prat C, Mitrophanous KA, Kingsman SM, Mazarakis ND, (2006) "Lentivirus-mediated gene transfer to the central nervous system: Therapeutic and research applications." Hum. Gene Ther. 17: 1–9.
• Butler SL, Johnson EP, Bushman FD, (2002) "Human immunodeficiency virus cDNA metabolism: Notable stability of two-long terminal repeat circles." J. Virol. 76: 3739–3747.

Light Induced Protein-Protein Interaction System.

The ability to control protein interactions inside living cells is important for investigations in a number of important biological processes including receptor binding, second-messenger activation, signal transduction systems, activation of G protein coupled receptors and the like. But current systems for monitoring or affecting protein interactions are cumbersome or require caged compounds with UV light induction. Recent work from the laboratory of Dr. Ricardo Dolmetsch and co-workers at the Department of Neurobiology at Stanford University Medical School have developed a light inducible system that utilizes two plant proteins involved in plant flowering to affect protein interactions in living mammalian cells. The two proteins, FKF1 and GI naturally bind during flowering. First FKF1 covalently binds FMN (flavin mono-nucleotide) through a sulfhydrile on the FKF1, upon light irradiation at 450 nm. Subsequently, the FMN-FKF1 complex binds exclusively to G1. Although G1 typically hydrolyses the cyteine-flavin bond, the half life of this activity is slow (several hours) inside mammalian cells. Using mCheery and YFP (red and yellow fluorescent proteins, respectively) the group was able to monitor binding of co-localization and membrane recruitment of the small G-protein Rac1 to a membrane bound farnesylated K-Ras protein. They were also able to show activation of a luciferase marker gene upon LAD (light-activated-dimerization) of a GI-Gal4 construct to a FKF1-VP16 construct using a UAS-luciferase marker gene. Although there are several technical hurtles that still need to be improved, the implications for this system of light activated dimerization of proteins of interest inside living cells is significant, and may greatly increase the number of cellular events that can be controlled by light illumination. For more information about these new systems, please visit our website or see the references below:

• Yazawa M, Sadaghiani AM, Hseuh B, Dolmetsch RE (2009) "Induction of protein-protein interactions in live cells using light." Nature Biotechnol. 27(10): 941-945.
• Sawa M, Nusinow DA, Kay SA, Imaizumi T (2007) "FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis." Science 318: 261-265.
• Nelson DC, Lasswell J, Rogg LE, Cohen MA, Bartel B, (2000) "FKF1, a clock-controlled gene that regulates the transition to flowering in Arabidopsis." Cell 101: 331-340.
• Mullins FM, Park CY, Dolmetsch RE, Lewis RS (2009) "STIM1 and calmodulin interact with Orai1 to induce Ca+2-dependent inactivation of CRAC channels" doi/10.1073PNAS 0906781106

Red Luminescent Firefly Luciferase Vectors.

An underlying theme that has guided advances in marker gene engineering has been that, all other things being considered, "the more red the better". This is largely due to the fact that longer-wavelength light is less phototoxic to cells and there are decreases in autofluorescence and scattering at these wavelengths. In addition, tissues are more transparent about the so-called "therapeutic window" of about 650 nm. Recently, researchers at Marker Gene have developed a new luciferase gene by utilizing point mutation and codon optimization of the luciferase sequence from the natural luciferase gene isolated from Luciola cruciata (the Japanese firefly). This novel recombinant DNA has been incorporated into vectors (M1394 and M1395) containing the highly expressing CMV and SV40 promoters for high expression activities in mammalian cells. The gene codes for an improved amino acid sequence which exhibits long-wavelength light emission (red color, EM 619 nm), as well as improved thermostability and higher expression levels in mammalian cells compared with other luciferases including the native luciferase derived from the American firefly Photinus pyralis. The expressed enzyme uses the same substrate, D-Luciferin (M0237), that is used with other luciferases, and activity can easily be detected using our convenient MarkerGeneTM Live Cell Luciferase Assay Kit (M0626). Additional substrates for use in detecting cloned activity in living tissues and in vivo include the membrane permeant D-Luciferin, ethyl ester (M0906). This makes these new vectors ideal candidates for use in vivo detection methods of cloned luciferase activity. It can also be used concurrently with vectors such as the pGL3 Photinus pyralis vectors for multiplexed detection of two cloning events in the same cell line.

Transfection of the vector into eukaryotic cells may be mediated by cationic lipid compounds like LipofectamineTM, calcium phosphate, DEAE-dextrans, or electroporation. Similar expression vectors are under construction for use in expression of this luciferase protein in both bacterial and plant cells.  Work is also currently underway to isolate the modified luciferase protein, which will be useful in coupled enzyme assays and general enzymology methods. Marker Gene has an open business model and will accept inquiries regarding licensing or manufacturing of this new luciferase for specific biological applications. For more information about these systems, please see the references below or visit our website.

• Tatsumi H, Masuda T, Kajiyama N, Nakano E (1989) "Luciferase cDNA from Japanese firefly, Luciola cruciata: cloning, structure and expression in Escherichia coli." J. Biolumin. Chemilumin. 3(2):75-78.
• Masuda T, Tatsumi H, Nakano E (1989) "Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata." Gene. 77(2):265-270.
• de Wet JR, Wood KV, Helinski DR, DeLuca M (1985) "Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli." Proc Natl Acad Sci U S A. 82(23):7870-7873.
• Mamaev SV, Laikhter AL, Arslan T, Hecht SM, (1996) "Firefly Luciferase: Alteration of the Color of Emitted Light Resulting from Substitutions at Position 286."
• Kajiyama, N, Nakano E, (1991) "Isolation and characterization of mutants of firefly luciferase which produce different colors of light." Protein Eng. 4: 691.
• Kajiyama N, Nakano E, (1993) "Thermostabilization of Firefly Luciferase by a Single Amino Acid Substitution at
  Position 217." Biochemistry 32: 13795-1 3799.

Compare Our Quality

CONTRACT RESEARCH @ markergene.com

• 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.

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.