Marker Gene Monthly Newsletter   

January, 2007

Volume 7, 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.

Lysosomal Acid Lipase Deficiency in Wolman’s Disease and Atherosclerosis.

Most lipids circulate in the body in the form of lipoprotein complexes. Simple, unesterified fatty acids are usually bound to serum albumin or other proteins in blood plasma, but phospholipids, triacylglycerols, cholesterol, and cholesterol esters are transported in the form of lipoproteins.  At various target sites, particularly in the capillaries of muscle and adipose cells, these lipoprotein particles are degraded by lipoprotein lipase, which hydrolyzes triacylglycerols.  Lipase action causes progressive loss of triacylglycerol (and apoprotein) and makes the lipoproteins smaller. This process gradually converts very low density lipoprotein (VLDL) particles to low density lipoprotein (LDL) particles, which are either returned to the liver for reprocessing or redirected to adipose tissues and adrenal glands. In a 24 hour period, nearly half of all circulating LDL is removed from circulation in this way.  LDL binds to specific LDL receptors, which cluster in domains of the plasma membrane known as coated pits.  These domains eventually are internalized in the cell to form coated vesicles which then fuse with lysosomes, and are degraded by lysosomal acid lipases.  Deficiencies in lysosomal acid lipase levels are the cause of a variety of serious disorders including atherosclerosis, which is one of the major causes of morbidity and mortality in the western world.  Lysosomal acid lipase (LAL) is the only hydrolase connecting extra-cellular with intra-cellular lipid metabolism for cleavage of triacylglycerols and  cholesteryl esters delivered to the lysosomes.  Patients with a deficiency of LAL show an accumulation of these lipids in the cells and develop pre-mature atherosclerosis.  In addition, Wolman’s Disease is a genetic abnormality caused by reduced levels of lysosomal acid lipase, and subsequent accumulation of lipids in lysosomes (a lysosomal storage disease).     Lysosomal acid lipase (LAL) is an essential enzyme for hydrolysis of both triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Its deficiency produces the human phenotypes for both Wolman disease (WD) and cholesteryl ester storage disease (CESD).    Measurement of acid lipase activity, in vitro and in vivo, is therefore of great interest for research into potential treatments of these important diseases.  Marker Gene has developed several new products for analysis of lipase activity in live cells including the MarkerGene^TM Fluorescent Lipase Assay Kit (M0612), MarkerGene^TM Long Wavelength Fluorescent Lipase Assay Kit (M1214), 1,2-Dioleoyl-3-(pyren-1-yl) decanoyl-rac-Glycerol (M0258), Resorufin Oleate (M1208) and 4-(Trifluoromethyl) umbelliferyl oleate (M0522).  Each of these substrates and kits have specific advantages for particular lipase analyses.  For more information, please see the references below or visit our website.  

• Merkel M., Tilkorn A.C., Greten H., Ameis D. (1999) ” Lysosomal acid lipase. Assay and purification.” Methods Mol Biol. 109:95-107. 
• Zschenker O.,  Illies T.,  Ameis D., (2006) “Overexpression of lysosomal acid lipase and other proteins in atherosclerosis.”  J Biochem (Tokyo) 140(1): 23-38. 
  
• Salvayre R ; Negre A ; Maret A ; Radom J ; Douste-Blazy L  (1987) “Extracellular origin of the lipid lysosomal storage in cultured fibroblasts from Wolman's disease.”  Eur. J. Biochem. 170(1-2): 453-8.
• Koster JF, Vaandrager H, van Berkel TJ. (1980) "Study of the hydrolysis of 4-methylumbelliferyl oleate by acid lipase and cholesteryl oleate by acid cholesteryl esterase in human leucocytes, fibroblasts and liver." Biochim Biopys Acta, 618(1): 98-105.
• Kuriyama M, Yoshida H, Suzuki M, Fujiyama J, Igata A. (1990) "Lysosomal acid lipase deficiency in rats: lipid analyses and lipase activities in liver and spleen." J Lipid Res, 31(9):1605-1612.
• Dousset, N., Negre, A., Salvayre, R., Rogalle, P., Dang, Q.Q., Douste-Blazy, L. (1988) "Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase." Lipids 23: 605-608.
  
• Negre, A., Salvayre, R., Dousset, N., Rogalle, P., Dang, Q.Q., Douste-Blazy, L. (1988) "Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice." Biochim. Biophys. Acta 963: 340-348.
  
• Hendrickson, H.S. (1994) "Fluorescence-bases assays of lipases, phospholipases, and other lipolytic enzymes."Analyt. Biochem. 219: 1-8.
  
• Müller, G., Jordan, H., Jung, C., Kleine H., Petry S., (2003) “Analysis of lipolysis in adipocytes using a fluorescent fatty acid derivative”  Biochimie 85(12): 1245-56.
• Beisson F., Arondel V., Verger R., (2000) “ Assaying Arabidopsis Lipase Activity”  Biochem. Soc. Trans. 28(6): 773-5
  

Mitochondrial ROS Staining with Dihydroethidium.

Dihydroethidium (M1241) (also called hydroethidium or hydroethidine) is the chemically reduced form of the commonly used DNA intercalating dye ethidium bromide (B-ring reduction).  This reduced dye is therefore very useful for detection of oxidative activities in viable cells, including respiratory burst in phagocytes, superoxide generation in mitochondria or as a vital stain in flow cytometry for imaging and analysis of intact cells.  It has also been shown to exhibit increased fluorescence in various models of apoptosis.  Dihydroethidium itself shows a blue fluorescence (absorption/emission: 355/420nm) in cell cytoplasm until oxidization to form ethidium which becomes red fluorescent (absorption/emission: 518/605 nm) upon DNA intercalation.  Only once it is internalized and dehydrogenated (oxidized) to ethidium, can it intercalate into DNA.  Because of their compromised membranes, only dead cells are typically labeled by ethidium bromide when it selectively binds to DNA.  But dihydroethidium is a neutral probe and is able to penetrate the cell membrane of live cells, staining their cytoplasm blue as well as the chromatin/nucleus of living cells red.  Dihydroethidium typically exhibits a uniform labeling of cells within 30-40 minutes.  Cell lysis of dihydroethidium-labeled cells can best be accomplished using Triton X-100 containing buffers.  In addition, Dihydroethidium has been incorporated into high-throughput assays to measure the effect of secondary agents or drugs on ROS activity in a live cell format.  Marker Gene now provides this important live cell stain for your assay development.  Samples of dihydroethidium should be stored under nitrogen or argon, to prevent autooxidation, and kept from light, especially when in solution.  For more information about this new live cell labeling probe, please see the references below or visit our website.

• Saiki I., Bucana C.D., Tsao J.Y., Fidler I.J. (1986) “Quantitative fluorescent microassay for identification of antiproliferative compounds.”  J. Natl. Cancer Inst., 77(6): 1235-1240.
• Frey, T., (1997) “Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems.” Cytometry, 28(3): 253-263.
• Budd, S. L, et al., (1997) “Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells” FEBS Lett., 415(1): 21-24.
• Zuo L., Christofi F.L., Wright V.P., Bao S., Clanton T.L.(2004) "Lipoxygenase-dependent superoxide release in skeletal muscle." J. Appl. Physiol. 97: 661-8
• King A., Gottlieb E., Brooks D.G., Murphy M.P., Dunaief J.L. (2004) "Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells." Photochem Photobiol 79: 470-5
• Nielsen H.G., Hagberg I.A., Lyberg T. (2004) "Marathon running leads to partial exhaustion of ROS-generating capacity in leukocytes." Med. Sci. Sports Exerc. 36: 68-73.

Cellulase Determination in Arabidopsis thaliana Floral Tissues.

Cellulases are a family of enzymes that include ß-Glucosidases, endoglucanases, and exoglucanases.  These enzymes cleave the ß-1,4-D-glycosidic bonds that link the glucose units comprising cellulose.  In addition to being produced by plants, cellulase activity is found in many fungi and bacteria, including some plant pathogens.  Most animal cells are not known to produce cellulase; cellulolytic activity is often carried out in animals by symbionts.  However, recent evidence does suggest cellulase production in some animals, such as insects and arthopods.  The study of cellulase activity has many applications in plant molecular biology, agriculture, and manufacturing.  Cellulase is also becoming important in the development of alternative fuel sources, as glucose obtained from cellulose hydrolysis is easily fermented into ethanol. Activity of most cellulases can now be monitored using our new long wavelength fluorescent substrate, Resorufin Cellobioside (M1238), contained in our MarkerGene^TM Fluorescent Cellulase Assay Kit (M1245).  Upon enzymatic hydrolysis, the bright red fluorescent compound, Resorufin (M0202) is released and activity measurements are easily obtained in a microtiterplate based assay format. The kit contains enough substrate for 200 assays and control experiments (100 μL reaction volume) and also contains reference standards and a detailed protocol for use.  The graph below shows representative data for these assays.  Flowering buds from two mature Arabidopsis thaliana plants (strain CS-20) are removed (90 mg tissue) and ground to a fine powder in liquid nitrogen.  The powder is suspended in reaction buffer (200 μL) (M1245-001)and centrifuged (13000 rpm) for 10 minutes.  The supernatant is then collected and added in triplicate(50μL) to wells ona 96-well microtiter plate (clear, flat bottom) along with 0.5mM substrate reagent (50 μL/well)  prepared by diluting 5mM Resorufin Cellobioside stock (in DMSO) (M1245-002) 1:10 in reaction buffer.   Fluorescenceis recorded using a Perkin-Elmer HTS 7000BioAssay Reader, using 550nm excitation and595nm emission filters. Fluorescence readings were recorded for 120 minutes at 3-minute intervals and subtracted from blanks at each time point.  For more information, please see the references below or visit our website.

• Villena G.K., Gutiérrez-Correa M.  (2006)  “Production of cellulase by Aspergillus niger biofilms developed on polyester cloth.”  Letters in Applied Microbiology.  43:  262-268
  
• Zhang Q., Bai G., Yang W., Li H., Xiong H. (2006).  “Pathogenic cellulase assay of pine wilt disease and immunological localization.’ Biosci. Biotechnol. Biochem. 70(11): 2727-32
  
• Nakata T., Miyafuji H., Saka S.  (2006) “Bioethanol from cellulose with supercritical water treatment followed by enzymatic hydrolysis.” Appl. Biochem. Biotechnol. 129-132: 47 6-85
  
• Han S.J., Yoo Y.J., Kang H.S.  (1995) “Characterization of a Bifunctional Cellulase and Its Structural Gene.”  J. Biochem, 270(43):  26012-26019.
  Boschker H.T.S., Cappenberg T.E. (1994) "A sensitive method using 4-Methylumbelliferyl-beta-Cellobiose as a Substrate to Measure (1,4)-beta-Glucanase Activity in Sediments." Applied and Environmental Biology, 60(10): 3592-3596.
  
• Chernoglazov V.M., Jafarova A.N., Klyosov A.A. (1989) "Continuous photometric determination of endo-1,4-beta-D-glucanase (cellulase) activity using 4-methylumbelliferyl-beta-D-cellobioside as a substrate." Anal Biochem, 179(1): 186-189.
  
• Thayer D.W.(1978) “Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus.”  Journal of General Microbiology, 106(1) 13-8.
  
• Ferrari T., Arnison, P.  (1974) “Extraction and Partial Characterization of Cellulases from Expanding Pea Epicotyls.”  Plant Physiol.  54:  487-493.

New Distributor in Korea.

Marker Gene is pleased to announce the addition of Leehyo Biosciences, Ltd. as our new distributor in Korea and Northern Asia.  LeehyoBio joins the other members of our growing list of international distributors that include Axxora Biosciences in Europe, VWR Scientific Products and Fischer Biotech in the USA and Europe, Dakewe Biosciences in China, Daiichi Pure Chemicals,Ltd. in Japan and many others.  Please visit their websites for more information on special offers and shipping information in your area. 

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.