Three U.S. researchers were awarded the 2009 Nobel Prize in Physiology and Medicine for their work on the discovery of telomerase activity and telomeres. Professors Elizabeth H. Blackburn of the University of California, San Francisco, Carol W. Greider at Johns Hopkins University School of Medicine and Jack W. Szostak who is at Harvard Medical School share the prize for their research on the unique DNA sequence (repeats of TTAGGG) and enzyme modification mechanisms of telomeres, which serve to protect the ends of chromosomes from damage and degradation. As cells age, their telomere lengths shorten with each round of replication until, at a certain point, the cells enter into senescence, apoptosis and cell death. On the other hand, overexpression of telomerase activity leads to telomere length being maintained and a delay of cellular degradation and cell death. Cancer cells for example, are able to maintain their telomere lengths and avoid degeneration (providing immortalized cell lines). Certain inherited diseases such as chronic obstructive pulmonary disease (COPD), coronary heart disease or chronic myeloproliferative diseases are characterized by defective telomerase activity.

The methods used to test telomere length and telomerase activity include PCR analysis, metaphase chromosome spread analysis by FISH (fluorescence in-situ hybridization) assay using fluorescently labeled or biotin labeled reverse telomere probes (often PNA probes) or by using the telomere repeat amplification protocols (TRAP assays) that employ a synthetic telomere primer and RT-PCR to monitor telomerase activity. For more information about these assays and methods, please see the references below. Marker Gene provides several new assay kits for use with telomerase activity assays, including the Fluorescence Microplate and Cellular Senescence Assay kits (M1389 and M1405), and Fluorescence Apoptosis Assay kits (M0815 - M0838). In addition, Marker Gene is developing a new series of direct FISH assays for metaphase chromosome spread analysis of telomere length and activity. For more information about these new products, please visit our website.

• Szostak JW, Blackburn EH, (1982) "Cloning yeast telomeres on linear plasmid vectors." Cell 29:245-255.
• Greider CW, Blackburn EH, (1985) "Identification of a specific telomere terminal transferase activity in Tetrahymena extracts." Cell 43:405-413.
• Greider CW, Blackburn EH, (1989) "A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis." Nature 337:331-337.
• Kim NW, Piatyszek MA, Prowse KR (1994) "Specific association of human telomerase activity with immortal cells and cancer." Science 266: 2011-2015.
• Viera A, Parra MT, Rufas JS, Suja JA, (2002) "Size heterogeneity of telomeric DNA in mouse meiotic chromosomes." Cytogen. Genome Res. 98: 221-224.

ROS Activity in Alzheimer's Disease

Alzheimer's disease is a degenerative neurological disorder that affects about 5 million people in the US, resulting in memory loss and dementia. One theory on the cause of Alzheimer's involves overproduction of amyloid beta proteins by neurons that has a multitude of effects including generation of reactive oxygen species (ROS), which in turn activate stress pathways in the neurons affected. Recent work from the laboratory of Professor Sandra Rossie at the Department of Biochemistry, Purdue University found that increases in the amount of a phosphatase enzyme PP5 in rat neural cells resulted in less cell death associated with reactive oxygen species activities. Conversely, decreasing PP5 was found to increase neuronal cell death. PP5 is a ubiquitously expressed serine/threonine phosphatase that is involved in the MAPK pathway. Overexpression of the protein phosphatase 5 (PP5) was found to prevent neuronal death by amyloid beta and shuts off various stress pathways. In addition, PP5 was also active against treatment with other ROS activating compounds, like H₂O₂. This data suggests that PP5 can protect neurons from cell death induced by reactive oxygen species produced by a variety of mechanisms, and not just the presence of amyloid beta. Their analyses used dihydroethidium (M1241) to measure ROS activity, the MTT assay to measure cell viability, and propidium iodide (M0793, M0795) staining to measure cell death.

The implications for potential therapeutic treatments that could increase PP5 activity and help prevent the loss of neurons that have excess amyloid beta protein plaques is significant. In addition, oxidative stress also plays a major neurotoxic role in other neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, and ALS (amyotrophic lateral sclerosis). For more information about these new systems please visit our website or see the references below.

• Sanchez-Ortiz E, Hahm BK, Armstrong DL, Rossie S, (2009) "Protein phosphatase 5 protects neurons against amyloid-beta toxicity." Journal of Neurochemistry 111(2): 391-402.
• Gong CX, Liu F, Wu G, Rossie S, Wegiel J, Li L, Inge, Iqbal G, Iqbal K, (2004) "Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5." Journal of Neurochemistry 88(2): 298-310.
• Liu F, Iqbal K, Grundke-Iqbal I, Rossie SS, Gong CX (2005). "Dephosphorylation of tau by protein phosphatase 5: impairment in Alzheimer’s disease." J. Biol. Chem., 280(3): 1790-1796.
• Chen L, Liu L, Yin J, Luo Y, Huang S, (2009) "Hydrogen peroxide induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway."
  Int. J. Biochem. Cell Biol. 41: 1284–1295.
  

A New High-Throughput alpha-Glucosidase Assay.

Pompe Disease is an autosomal recessive genetic disorder that affects approximately 1 in 40,000 live births. Patients with Pompe Disease suffer from progressive muscle weakness, impaired mobility and respiration, and the most severe cases have devastating cardiomegaly, hypotonia and hepatomegaly with death usually occuring before age 2 years due to cardiorespiratory failure.

Pompe Disease, also known as acid maltase deficiency (AMD) or glycogen storage disease type II (GSD-II), was found to be caused by a mutation in the enzyme alpha glucosidase. This causes accumulation of excess glycogen in the lysosomes of many cells in the body, including muscle and neuronal cells. Interestingly Pompe disease was the first lysosomal storage disorder (LSD) to be identified, from a family of more than 40 diseases that interfere with the body's ability to degrade complex molecules within the lysosome. But clinical treatments for this disease are still lacking. Small molecule chaperone proteins that bind to the enzyme and correct the misfolding and mistrafficking of the mutant enzyme have emerged as a new therapeutic approach for these lysosomal storage disorders. In addition, direct alpha glucosidase enzyme replacement therapy has emerged as a therapeutic target for Pompe as well as type II diabetes, and a variety of glucosidase inhibitors that can activate the chaperone proteins have been used in the clinic as alternative treatments for this disease.

Recent work from the laboratory of Dr. Ellen Sidransky and coworkers at the Human Genome Research Institute at the National Institutes of Health have developed a new assay for the alpha-glucosidase enzyme based upon a new substrate resorufin alpha-D-glucopyranoside. This substrate, upon enzyme activity, produces the red fluorophore resorufin, with emission at 590 nm. In addition, because the pKa value of the enzyme reaction product, resorufin (pKa 6.0), is significantly lower than other fluorophores, it enables continuous kinetic assay at the lower physiological pH values present in the lysosome (pH 5-6). In the search for new therapeutics by high throughput screening of compound libraries, this new enzyme assay with the red fluorogenic substrate can avoid or minimize false positives due to the significant amount of drug compounds having absorption or even emission in the blue wavelength regions found with most other common fluorogenic substrates. Thus, this new resorufin alpha-D-glucoside substrate was found to be a better assay choice for the identification of new activators and inhibitors using HTS. For more information about these new assays, please see the references below:

• Motabar O, Shi ZD, Goldin E, Liu K, Southall N, Sidransky E, Austin CP, Griffiths GL, Zheng W (2009) "A new resorufin-based a-glucosidase assay for high-throughput screening." Analytical Biochemistry 390:79-84.
• Hirschhorn R, Arnold J. J. Reuser AJJ, :"Glycogen Storage Disease Type II: Acid Alpha-glucosidase (Acid Maltase) Deficiency." In: Scriver C, Beaudet A, Sly W, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th Edition. New York: McGraw-Hill, 2001. 3389-3420.
• Slonim AE, Bulone L, Ritz S, Goldberg T, Chen A, Martiniuk F, (2000) "Identification of two subtypes of infantile acid maltase deficiency." J. Pediatr. 37(2):283-5.
• Chen YT, Amalfitano A, (2000) "Towards a molecular therapy for glycogen storage disease type II (Pompe disease)." Mol. Med. Today 6(6): 245-51.
• Coleman DJ, Studler MJ, Naleway JJ, (2007) "A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-b D-cellobioside. " Anal.Biochem.371:146 -153.

DNA Fluorescence Labeling Sets.

One of the most common fluorescent dye labeling sets for DNA sequencing and PCR amplification is the four-dye set including FAM, JOE, TAMRA and ROX. This dye set contains 6-carboxyfluorescein (FAM), 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein (JOE), 6-carboxytetramethylrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX), most commonly as their NHS ester reactive forms. The excitation and emission wavelengths for these dyes show little overlap (see the figure at left) with EX/EM 494/517; 528/554; 560/583 and 587/607nm respectively. These dyes are commonly used in dye-terminator sequencing methods by attachment to dideoxynucleotide triphosphates (ddNTP's). But they can also be used in other DNA or RNA labeling methods.

This dye set is still used in Applied Biosystems automated sequencing systems including the ABI 373 and ABI 377 series instruments. These dyes are routinely chemically introduced into primers or as fluorescently labeled nucleoside triphosphates and are then incorporated either using PCR amplification or by using DNA polymerases, RNA polymerases or terminal polynucleotide transferase. Marker Gene now provides these dyes and their reactive NHS ester forms in high-purity form (M1472, M0962, M0972, M0984). For more information about these dyes and labeling methods, please contact our technical assistance department, see the references below or see our website.

• Mackay IM (2004) "Real-time PCR in the microbiology laboratory." Clin. Microbiol. Infect. 10(3): 190-212
• Ju J, Ruan C, Fuller CW, Glazer AN, Mathies RA (1995) "Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis." Proc. Natl. Acad. Sci. USA (92):10 4347-4351.
• Ansorge W, Posenthal A, Sproat B, Stegemann J, Voss H, (1988) "Non-radioactive automated sequencing of oligonucleotides by chemical degradation." Nucleic Acids Res. 16: 2203-2206.
• Prober JM, Trahnor SH, Dann RJ, Hobbs FW, Robertson CW, Zagursky RJ, Cocuzza AJ, Jensen MA, Baumeister K (1987) "A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides." Science 238: 336-341.
• Smith LM, Fung S, Hunkapiller MW, Hunkapiller TJ, Hood LE (1986) "The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis." Nucleic Acids Res. 13: 2399-2412.

New MGT Website @markergene.com.

Marker Gene has unveiled its new customer Web site today at www.markergene.com.   The site is designed to help consumers and researchers get all the latest information on new marker and reporter gene technologies, assay reagents and kits as well as product information in one convenient place. The new site will feature information from all areas of research and industrial use across the spectrum of product categories with critical protocol and assay development information, including updated laboratory protocols, alerts on new methods with literature references, and the latest news from the key research in the field. Consumers can sign up in one easy place to receive email and Newsletter alerts on new methods and hear about new developments from the top scientific experts in the field of marker gene applications and technologies.  Later phases of the site to be launched will include feeds for texting and mobile phones. The site will also feature a widget that the public and the media will be encouraged to download and promote on their Web sites as well as upcoming scientific networking sites. The widget will instantly update viewers with the latest product information and advances and will be a valuable research and development tool. Please visit our new website now and tell us your opinion or help us with our constant improvements.

Compare Our Quality

Marker Gene strives to offer our customers products of the highest quality at competitive prices. Our years of experience allow us to provide excellent products in a timely manner. For more information, visit our website at http://www.markergene.com/ and click on the "PRODUCTS" link . We think you will appreciate our efforts to maintain excellent quality in our items for your research. For more information about any of our products, simply call 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 information 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 (CRO) 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.

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.