Marker Gene Monthly Newsletter   

March, 2006

Volume 6, Number 3

© 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.

Lysosomal Storage Disease Detection.

The lysosomal storage diseases are a family of genetic human metabolic diseases that, in their severest forms, cause mortality as a result of progressive neurodegeneration. They are caused by mutations in the genes encoding glycohydrolase enzymes that catabolize glycosphingolipids within the lysosome.  When there is such a lysosomal enzyme deficiency, the deficient enzyme's undegraded substrates gradually accumulate within the lysosomes causing a progressive increase in the size and number of these organelles within the cell. This accumulation within the cell eventually leads to malfunction of the organ and to the gross pathology of a lysosomal storage disease, with the particular disease depending on the particular enzyme deficiency.  Among the most common of these diseases are Gaucher’s disease caused by a lack in the normal form of the enzyme glucocerebrosidase causing an accumulation of glucocerebroside glycolipids. The fluorogenic substrate fluorescein di-b-D-glucopyranoside (FDGlc, M0881) is one of the most sensitive substrates for detecting general -glucosidase activity in cell extracts.  Marker Gene also provides the red fluorogenic resorufin b-D-glucopyranoside (M0569) substrate, which is a more stable, long-wavelength analog for detection of glucosidase activities.  Sandhoff’s disease and the more severe Tay-Sachs syndrome are caused by a defects in Hexosaminidase A/B enzymes and accumulation of several types of gangliosides inside lysosomes.  Marker Gene has introduced a new substrate resorufin b-D-N-Acetylgalactosamine (M1037) for analysis of these enzymes from cell extracts.  Finally, Krabbé disease is caused by a deficiency of galactocerebrosidase, an essential enzyme for myelin metabolism.   Deficiency of this enzyme causes accumulation of galactocerebrosides in various tissues.  Marker Gene provides several sensitive substrates for measuring levels of galactocerebrosidases including 4-Methylumbellifery-b-D-Galactopyranside (MUG, M0241), 4-Trifluoromethylumbelliferyl-b-D-Galactopyranoside (TFMU-Gal, M0252), Fluorescein di-b-D-Galactopyranoside (FDG, M0250) and Resorufin-b-D-Galactopyranoside (Res-Gal, M0203).  For more information about these assays and methods for measuring the benefits of individual substrates, please see the references below, or visit our web site.

• van Es H.H., Veldwijk M., Havenga M., Valerio D. (1997) "A flow cytometric assay for lysosomal glucocerebrosidase" Anal. Biochem.247:268-271.
• Chan KW; Waire J; Simons B., (2004) "Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay." Anal Biochem  334(2): 227-33.
• Rudensky B., Paz E., Altarescu G., (2003)  "Fluorescent flow cytometric assay: a new diagnostic tool for measuring beta-glucocerebrosidase activity in Gaucher disease." Blood Cells Mol. Dis.  30(1): 97-9.
• Daniels L.B., Glew R.H., Diven W.F., Lee R.E., Radin N.S., (1981) “An improved fluorimetric leukocyte b-glucosidase assay for Gaucher's disease.” Clin. Chim. Acta 115: 369-375.
• Kaxpova E.A., Voznyi Ya V., Dudukina T.V., Tsvetkova I.V., (1991) “4-Trifluoromethylumbelliferyl glycosides as new substrates for revealing diseases connected with hereditary deficiency of lysosome glycosidases.” Biochem. Int.  24: 1135-1144.
  

Phosphodiesterase Analysis Techniques.

Phosphodiesterases catalyze the hydrolysis of phosphodiester bonds in nucleic acids and cyclic nucleotides.  Although phosphodiesterases have many important cellular roles, there are few effective methods to monitor their activity in real time with high sensitivity.  For example, nucleotide pyrophosphatase/phosphodiesterases (NPPs), one group of phosphodiesterases, have been reported to be implicated in the regulation of various intra- and extracellular processes, including cell differentiation and motility, bone and cartilage mineralization, and signaling by nucleotides and insulin. Some important proteins categorized in NPPs such as PC-1 7 and Autotaxin 8 have been the focus of much interest in recent years.  Phosphodiesterase I (EC 3.1.4.1) is one of the main NPP enzymes, and new probes for phosphodiesterase I activity are being developed.  Many of these new probes contain either fluorophore quencher pairs, or utilize fluorescence energy resonance transfer (FRET) in which the fluorescence of one fluorophore excites a second fluorophore in the substrate.  Berkessel and Riedl have developed fluorescence reporters for phosphodiesterase I activity, in which a naphthalene moiety acts as the fluorophore and an azobenzene group as the quencher.  Upon addition of phosphodiesterases these reporters fluoresce, but have limitations in biological applications because of their short excitation wavelength and weak fluorescence.  A ratiometric fluorescent substrate has recently been developed for phosphodiesterase activity by a group from the University of Tokyo and Chiba University, using a FRET pair with a chloroacetylcoumarin as a donor, and fluorescein as acceptor, and a phosphodiester as a linker.  This new substrate has increased sensitivity and can be used for real-time analysis of these important enzymes.  For more information about these new substrates, please see the references below, or visit our website.

• Takakusa, H., Kikuchi, K., Urano, Y., Sakamoto, S., Yamaguchi, K., Nagano T.,  (2002) “Design and Synthesis of an Enzyme-Cleavable Sensor Molecule for Phosphodiesterase Activity Based on Fluorescence Resonance Energy Transfer” J. Amer. Chem. Soc. 124(8): 1653-1657.
• Strater, N., Lipscomb, W. N., Klabunde, T., Krebs, B. (1996) Angew. Chem Int. Ed., 35: 2024-2055.
• Bollen, M., Gijsbers, R., Ceulemans, H., Stalmans, W., Stefan, C. (2000) Crit. Rev. Biochem. Mol., 35: 393-432.
• Zimmermann, H., Braun, N. (1999) Prog. Brain Res., 120: 371-385.
• Zimmermann, H. (1999) Trends. Pharmacol. Sci., 20: 231-236.
• Goding, J. W. (2000) J. Leukoc. Biol., 67: 285-311.
• Goding, J. W., Terkeltaub, R., Maurice, M., Deterre, P., Sali, A., Belli, S.I. (1998) Immunol. Rev., 161: 11-26.
• Stracke, M. L., Clair, T., Liotta, L. A. (1997) Adv. Enzyme Regul., 37: 135-144.
• Clair, T., Lee, H. Y., Liotta, L. A., Stracke, M. L. (1997) J. Biol. Chem., 272: 996-1001.
• Berkessel, A., Riedl, R. (1997) Angew. Chem Int. Ed. Engl., 36: 1481-1483.
• Kawanishi, Y., Kikuchi, K., Takakusa, H., Mizukami, S., Urano, Y., Higuchi, T., Nagano, T. (2000) Angew. Chem Int. Ed., 39: 3438-3440.

Fluorescence of Native GFP and Proteins and Peptides.

There are three aromatic amino acid residues (tryptophan, tyrosine, phenylalanine) that can contribute to intrinsic protein fluorescence. The fluorescence of a folded protein is a mixture of the fluorescence from these individual aromatic residues. Protein fluorescence is generally excited at between 280 nm to 295 nm and most of the emission is  due to tryptophan residues.  A special case of fluorescence occurs in Green Fluorescent Protein where the fluorophore originates from an internal serine-tyrosine-glycine sequence that is post-translationally modified to a 4-(p-hydroxybenzylidene)- imidazolidin-5-one structure.  Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with emission at 509 nm (green).  The GFP from the sea pansy exhibits a single major excitation peak at 498 nm. Although most small molecule fluorophores are quenched in the solid state, crystals of GFP exhibit a nearly identical fluorescence spectrum and lifetime to that found in aqueous solution.  Exciting wild type GFP at 395 nm leads to rapid quenching of the fluorescence with an increase in the 475 nm excitation band. This photoisomerization effect can be accomplished by irradiation of GFP with UV light.  In addition, changes in pH can also lead to a similar effect, reducing pH leads to reducing fluorescence using 395 nm excitation and increasing the 475 nm excitation.   A variety of mutants of the GFP gene have been produced that have increased fluorescence and the major excitation peak red-shifted to 490 nm with the emission staying at about 510 nm. These proteins are therefore better suited for use with standard optical filter sets.  For more information about native GFP fluorescence and it’s use, please see the references below or visit our website.

• Prescher J.A., Bertozzi C.R., (2005) “Chemistry in living systems.” Nature Chem. Biol., 1(1): 13-21.
• Ward, W.W., Bokman, S.H., (1982) “Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein.” Biochemistry 21(19): 4535-4540.
• Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. (1994) “Green fluorescent protein as a marker for gene expression.” Science 263: 802–5
• Tsien, R.Y., (1998) “The Green Fluorescent Protein” Annual Review of Biochemistry  67(1): 509-545.
• Jiskoot W., Hlady V., Naleway J.J., Herron J.N., (1995) ” Application of fluorescence spectroscopy for determining the structure and function of proteins.” Pharmaceutical biotechnology 7: 1-63.

Alkaline Phosphatase Analysis of Pluripotent Stem Cells.

Pluripotent stem cells are difficult to maintain because the can easily differentiate in culture.  The undifferentiated stem cell is characterized by a high level of alkaline phosphatase expression. Alkaline phosphatase assays can therefore be used to help determine if stem cells are undifferentiated or are beginning to differentiate. Methods to detect phosphatase activity in stem cell preparations include azo dye methods and X-Phos (BCIP/NBT) staining. A new method has been developed using a precipitating phosphatase substrate that is soluble and fluoresces weakly in the blue range in solution, but forms a bright yellow-green fluorescent precipitate upon phosphatase activity.  This precipitate has a large Stokes shift (>100nm) and can be used in dual or multicolor applications with other counterstains. This precipitate is also very photo stable. The excitation/emission of the fluorescent product is 345nm and 530nm respectively.  The staining pattern will appear yellow-green against a blue background using a Hoechst/DAPI longpass filter set.   The method has been used on several human and mouse embryonic stem cell lines (fixed cells), and allows efficient and convenient detection of alkaline phosphatase in embryonic stem cells. This fluorescence-based system can be used in conjunction with other stem cell markers to provide assessment of overall in vitro stem cell pluripotency.  For more information about these new assays, please visit our website or see the references below.

• Shamblott MJ et al. (1998) “Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA 95(23): 13726-13731.
• Plaia T.W., et al. (2004)Fluorescence-based analysis of embryonic stem cell pluripotency. ATCC Connection 24(2): 1 and 4,.
• Haugland R. P., Zhang; Y.Z, Yue S. T., Terpetschnig E., Olson N. A., Naleway J.J., Larison K. D., Huang; Z. (1994) “Enzymatic analysis using substrates that yield fluorescent precipitates”  US Patent 5,316,906.
• Naleway, J. J. , Fox, C. M. J. , Robinhold, D. , Terpetschnig, E. (1994) “Synthesis and use of new fluorogenic precipitating substrates”  Tet. Lett. 35(46): 8569.
  

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. 

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