Marker Gene Monthly Newsletter - Volume 11, Number 9 - September, 2011

ROS Measurement in Cardiomyocytes.

The constant stretching and relaxation of the heart muscle can cause production of reactive oxygen species (ROS) that are influential in cardiac health and disease. Even a small diastolic stretch can cause a burst of Ca+2 ion release from intracellular stores that is accompanied by local production of ROS. Recent work from the University of Maryland School of Medicine has developed an ingenious method of measuring both stretch-induced ROS activation as well as Ca+2 ion waves in cardiac myocytes simultaneously by attaching single cells to stiff glass rods using a biological adhesive, MyoTak, and then measuring ROS activity by staining with the fluorescent ROS sensor 2',7'-dichlorofluorescin diacetate (M0807) as well as the Calcium ion indicator Fluo-4-acetoxymethyl ester, with or without various inhibitors, or drug compounds. They were able to demonstrate increased ROS production and associated Ca+2 ion release in various disease models versus normal myocytes.

The ROS sensor 2',7'-dichlorofluorescin diacetate (M0807) has been a widely documented probe for monitoring oxidative stress in cellular studies. The esterified form of the dye (2',7'-dichlorofluorescin diacetate) rapidly penetrates cell membranes and becomes deacetylated by intracellular esterases. The resulting nonfluorescent dichlorofluorescin is then trapped in the cytosol and, upon oxidation, is converted to the highly fluorescent dichlorofluorescein dye, serving as a sensitive cytosolic marker for oxidative stress. In tissue samples, similar measurement of ROS species could be monitored using dihydroethidium (hydroethidine, M1241). For more information about these techniques, please see the references below or visit our website

• Benjamin L. Prosser BL, Christopher W. Ward CW, W. J. Lederer WJ (2011) "X-ROS Signaling: Rapid Mechano-Chemo Transduction in Heart." Science 333(6048): 1440-1445.
• Williams IA, Allen DG, (2007) "The role of reactive oxygen species in the hearts of dystrophin-deficient mdx mice." Am. J. Physiol. Heart Circ. Physiol. 293: H1969.
• Luther M. Swift LM, and Narine Sarvazyan N, (2000) " Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress." Amer. J. Physiol. Heart Circ. Physiol. 278:982-990.
• LeBel CP, Ischiropoulos H, Bondy SC (1992) "Evaluation of the probe 2,7-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress." Chem. Res. Toxicol. 5:227–231.
• Zhu H, Bannenberg GL, Moldeus P, Shertzer HG (1994) "Oxidation pathways for the intracellular probe 2,7-dichlorofluorescein." Arch. Toxicol. 68:582–587.
  

Fluorogenic Pyrosequencing Technique.

Typical pyrosequencing methods utilize the released PPi generated when each base is added to a growing nucleic acid chain along with the enzymes ATP sulfurylase and firefly luciferase to generate a luminescent signal for detection in a high-throughput sequencing protocol. However, this method produces a transient light signal, while fluorescent techniques are able to produce a steady signal that can be analyzed over longer periods or repeatedly. Recent work from the laboratory of Dr. Xioliang Sunney Xie and coworkers at Harvard University has developed a new fluorogenic sequencing technique that utilizes polydimethylsiloxane (PDMS) microcells and fluorogenic (3'-O-methyl-5(6)-carboxyfluorescein) nucleotide tetraphosphate substrates for analysis. These extended gamma-phosphate esters were utilized to increase the distance between the dye and polymerase active site, recovering the activity of standard polymerase reaction kinetics. The PDMS microwell reactors reduced unwanted diffusion of the released dyes into the reaction wells and loss of signal. The combined method was able to image 30 base reads with 99% raw accuracy. This new real-time monitoring method has potential application for microfluidic integration or the ability to sequence raw biomaterial samples. To find out more about these methods, please see references below or visit our website.

• Sims PA, Greenleaf WJ, Duan H, Xie XS (2011) "Fluorogenic DNA Sequencing in PDMS Microreactors." Nature Methods 8(7) 575-579.
• Ronaghi M, Karamohamed S, Pettersson B, Uhlen M, Nyren P, (1996) "Real-Time DNA Sequencing Using Detection of Pyrophosphate Release." Anal. Biochem. 242: 84–89.
• Baback Gharizadeh, Tommy Nordstrom, Afshin Ahmadian, Mostafa Ronaghi, Pål Nyren (2002) "Long-Read Pyrosequencing Using Pure 2'- Deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-Isomer." Anal. Biochem. 301: 82–90.
• Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer MLI, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Pengguang Yu M, Begley RF, Rothberg JM (2005) "Genome sequencing in microfabricated high-density picolitre reactors." Nature 437: 376-380.
  

Potential Repair Mechanism for Pompe Disease.

Lysosomal Storage Diseases occur due to genetic defect in one or more lysosomal enzymes and a resulting functional failure of cells to process lysosome contents in normal metabolism. As a result, lysosomes accumulate in tissues and affect normal development. Lysosomal Disorders have recently been implicated in a number of diseases including Parkinson's, Krabbe, Gaucher, Tay-Sachs, Huntington's, Pompe and Sanfillipo. Recent work from the laboratories of Dr. Andrea Ballabio and coworkers at the Telethon Institute of Genetics and Medicine, Italy as well as the NIH-NHBLI, Baylor College of Medicine, Texas Childrens Hospital and the Department of Pediatrics at Federico II University, Italy has identified a transcription factor EB (TFEB), which is a bHLH-leucine zipper transcription factor that regulates lysosomal exocytosis. TFEB was found to increase the pool of lysosomes that could fuse with the Plasma Membrane (PM) and thus be exocytosed. By overexpression of TFEB they were able to restore normal cellular morphology in a cellular model of Huntington's disease as well as the phenotype of cells from a murine model of Parkinson's disease and human glycogenosis type II Pompe disease. These results represent a potential alternative therapeutic option for these devastating diseases of early childhood and adults. For more information about these techniques, please see the references below or visit our website

• Medina DL, Fraldi A, Bouche V, Annunziata F, Mansueto G, Spampanato C, Puri C, Pignata A, Martina JA, Sardiello M, Palmieri M, Polishchuk R, Puertollano R, Ballabio A, (2011) "Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance." Develop. Cell 21: 421–430.
• Sardiello M, Palmieri M, di Ronza A, Medina DL, Valenza M, Gennarino VA, Di Malta C, Donaudy F, Embrion V, Polishchuk RS, (2009) "A gene network regulating lysosomal biogenesis and function." Science 325, 473–477.
• Dehay B, Bove J, Rodrıguez-Muela N, Perier C, Recasens A, Boya P, Vila M (2010) "Pathogenic lysosomal depletion in Parkinson’s disease." J. Neurosci. 30, 12535–12544.
  

RubyGlow^TM Bacterial Detection Kit.

Rapid and accurate measurement of viable bacterial cell number and bacterial cell growth is required in a variety of biological applications. Traditional assessment of bacterial contamination or viability has been made via plate assay or using chromogenic or fluorogenic substrates.  An alternative technology used in quantitating all of these parameters including cell number, proliferation, growth, viability or cytotoxicity is based on the generation of ATP in metabolically active cells. Studies have shown that the level of intracellular ATP per cell is highly regulated and remains essentially constant in a cell population.  The cell responds to the increased demand for ATP by increasing the production or turnover rate of ATP. Increasing levels of ATP are therefore a reflection of cell proliferation and an increase in the number of viable cells per well.  Controlled studies have shown that ATP measurements correlate well with traditional methods.  Any form of cell injury results in a rapid decrease in ATP levels; thus, measuring ATP in drug treated cells can assess the viability of the cells as well as cytotoxicity of the drug.

The most sensitive technique for ATP measurement has proven to be the luciferin-luciferase bioluminescent assay.  The use of the luciferase enzyme has become highly valuable as a genetic marker gene due to the convenience, high sensitivity and linear range of the luminescence assay.  The reaction involved is described as:

Luciferase + ATP + D-Luciferin + O₂  ->  Oxyluciferin + AMP + PP_i + CO₂ + Light.

Thus measuring the units of light output will accurately reflect the amount of ATP in a sample or reaction. Using genetic engineering, we have generated a new luciferase which exhibits long-wavelength light emission, as well as improved thermostability, compared to the native firefly luciferase often used in these assays.  The light output is stable over several hours and thus eliminates the necessity for substrate injection.  We have adapted this new luciferase into a series of kits for use in a microtiter plate format and thus made them amenable to high-throughput applications.  Our new RubyGlow^TM Luminescent Bacterial Detection Kit (Product M1573) can detect bacteria from a number of media or sources with an extremely high sensitivity (low detection limit).  In addition, extended incubation (2-4 hours) of samples at 37 ºC can significantly lower the detection limit and sensitivity of detection using a protocol that can be completed in one day. Data variation is low and confidence is high compared with several current “Add and Read” commercial kits.  Due to its stable light emission, we have adapted this kit for use in a microtiter plate format; this enables the processing of a large number of samples (such as food or clinical samples) at the same time.  This microplate format assay is amenable to high throughput (HTS) applications for analysis of bacterial susceptibility and antibiotic screening. This new kit adds to our line of RubyGlow^TM Luciferase kits (M1574, M1575 and M1576) that produce a red light emission (EM 619 nm) upon luciferin conversion, while most of the commercially available luciferase kits still use older green light emitting (562 nm) firefly analogs.  This unique feature enables the possibility of multiplexing.  The new kit components contain no radioisotopes, are not toxic to cells and are environmentally safe.  The kits provide enough reagents and a detailed protocol sufficient for 100 reactions in 96-well microplate format.  For more information about these new kits, please see the references below, or visit our website.

• Cunningham BA. (2001) “A growing issue: cell proliferation assay” The Scientist 15(13): 26.
• 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." J. Amer. Chem. Soc. 118: 7243-7244.
• 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." Biochem 32: 13795-13799.
• Crouch SP, Kozlowski R, Slater KJ, Fletcher J. (1993) "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity," J. Immunol. Methods 160(1): 81-88.

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