Reactive Oxygen Species Levels Related to Circadian Rhythms.

In healthy aerobic cells, reactive oxygen species (ROS) generation occurs at a controlled rate, but, under high stress conditions, its production can be greatly increased, resulting in changes of many cell components including proteins and lipids. There is increasing evidence that circadian rhythms may contribute to synchronization of physiology and cellular metabolism that affect the health of cells and organisms, inhibiting certain pathologies such as cancer and premature aging by an anti-oxidation defense mechanism. Recently work from the laboratory of Professor Jadwiga Giebultowicz and colleagues at the Department of Zoology at Oregon State University have supported this theory by measuring the effect of ROS induction in relation to null mutation gene knockouts in the core clock gene period (per) which abrogated circadian rhythym clock function in the fruit fly Drosophila melanogaster. This gene knockout increased susceptibility to hydrogen peroxide compared to wild-type flies, coinciding with enhanced
generation of mitochondrial H2O2 and decreased catalase activity due to oxidative damage. It was also found that the fruit flies had their greatest ability to manage oxidative stress in the early morning, shortly before they had to deal with the challenges of the day – and the least natural defense in late afternoon or evening, a time when DNA damage reached its peak. Measurement of mitochondrial ROS levels and catalase activity were performed using fluorescent analyses. The implications for timing in drug delivery, as well as disease effects from disrupted circadian cycles (swing-shift workers, jet-lag, etc.) may be important.

Marker Gene provides a variety of products and methods for measuring ROS species in live cell culture including Dihydroethidium , Dichlorofluorescin diacetate, Dihydrorhodamine 123 as well as the MarkerGeneTM Live Cell Fluorescent Reactive Oxygen Species Detection Kit (M1049).

Dichlorofluorescin diacetate (H2DCFDA, M0807) is a cell-permeant indicator for reactive oxygen species that is nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell.  The reduced substrate releases the highly fluorescent dye 2',7'-dichlorofluorescein and allows easy detection of peroxidase activity (oxidative metabolism, intracellular regulation of reactive oxygen species or oxidative burst) inside living cells.  When the cell begins to produce reactive oxygen species, the highly fluorescent dye 2',7'-dichlorofluorescein is produced, with EX: 495nm and EM: 529 nm.  Marker Gene now provides this reagent in an easy to use kit form in the MarkerGeneTM Live Cell Fluorescent Reactive Oxygen Species Detection Kit (M1049), that includes an inducer (for positive control applications), the H2DCFDA substrate, standards and a detailed protocol for quick and easy discrimination of oxidatively stressed and nonstressed cells by fluorescence microscopy, microtiterplate assay or photomicroscopy. 

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. For more information about these methods, please see the references below, or visit our website.

• Krishnan N, Davis AJ, Giebultowicz JM (2008) "Circadian regulation of response to oxidative stress in Drosophila melanogaster." Biochem. Biophys. Res. Commun. 374(2):299-303.
• Hardeland R, Coto-Montes A, Poeggeler B (2003) "Circadian rhythms, oxidative stress, and antioxidative defense mechanisms." Chronobiol Int. 20(6):921-62.
• Diaz G, Liu S, Isola R, Diana A, Falchi AM (2003) "Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes." Histochem. Cell Biol. 120: 319-25.
• Budd, S. L, (1997) “Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells” FEBS Lett., 415(1): 21-24.
• King A., Gottlieb E., Brooks D.G., Murphy MP, Dunaief JL (2004) "Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells." Photochem Photobiol 79: 470-5.
  
  

Light Induced K+ -ion Channel Control.

Eukaryotic Cells have ion channels on their surface that help establish and control voltage gradients and overall cell potential across their membranes by restricting or allowing the flow of ions. For neurons, these ion channels can also elicit physiological responses, and can trigger synaptic transmission events. Regulating the firing of neurons in response to external stimuli has been the the topic of neuroscience research for many years. Recently work from the laboratory of Professor Richard Kramer and colleagues at the Department of Molecular and Cell Biology at the University of California-Berkeley has developed a series of new photoactivatable affinity compounds that can control the K+-ion channels in neurons using only light stimulation. These new compounds, termed photoswitchable affinity labels (PALs) are small molecule compounds that bind to the K+-ion channels, and change conformation (are photoisomerizable) upon illumination with different frequencies of light. The PALs contain a quaternary ammonium that bind to the core of the K+-ion channels and block ion conduction, an electophilic group (such as an epoxide or chloroacetamide) that causes permanent /covalent binding to a nucleophilic amino acid side chain in the channel, and an azobenzene function that is photoisomerizable. Upon exposure to 380 nm light, the azobenzene iosmerizes to its shorter cis-form, while exposure to 500 nm light (or darkness) causes it to relax back to the trans-configuration. In the trans-configuration, the PAL blocks the K+-ion channel and causes depolarization of the membrane potential. Simple one second flashes of 380 nm light are sufficient to open the K+-ion channels and cause neuron firing. Although this type of neuronal transmission control has been attempted before with caged neurotransmitters or by genetic engineering, this method which can be used with endogenous proteins in many cell types, can conceivably be used to impart optical control of neurons in cells, tissue culture, or even in humans, using fiber optic methods. In addition the PAL-modification has not appeared to cause obvious deleterious effects on cell growth or morphology of cultured hippocampal neurons. For more information about these exciting new methods, please see the references below or visit our website.

• Fortin DL, Banghart MR, Dunn TW, Borges K, Wagenaar DA, Gaudry Q, Karakossian MH, Otis TS, Kristan WB, Trauner D, Kramer RH. (2008) "Photochemical control of endogenous ion channels and cellular excitability." Nat Methods 5(4):331-8.
• Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) "Light-activated ion channels for remote control of neuronal firing." Nat Neurosci. 7(12):1381-6.
• Knöpfel T (2008) "Expanding the toolbox for remote control of neuronal circuits." Nat Methods 5(4):293-5.
• Callaway EM, Yuste R (2002) "Stimulating neurons with light." Curr Opin Neurobiol. 12(5):587-92.
• Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH. (2004) "Light-activated ion channels for remote control of neuronal firing." Nat Neurosci. 7(12):1381-6.

New Carbohydrate Labeling Systems.

Nearly all native proteins contain post-transcriptional modification with glycosidic elaboration (sugars) whose structures are dependent both on species and cell type. The characterization of the complex oligosaccharides obtained from these glycoproteins has proven a difficult and time consuming endeavor. At the recent International Carbohydrate Symposium in Oslo, Norway, Marker Gene introduced an improved method of labeling and characterizing the carbohydrates derived from transgenic Arabidopsis thaliana species. The protocol involves analyzing these carbohydrates through covalent labeling with a fluorescent reagent (1,5-EDANS), and analysis by a combination of methods including PAGE, HPLC and 2-dimensional thin-layer chromatography (TLC). The principle involves removal of the oligosaccharides from native proteins by hydrazinolysis, enzymatic digestion or acidic hydrolysis with TFA followed by reductive amination of the reducing sugars and analysis of the resultant glycamines. The advantages of using the 1,5 EDANS fluorophore include its low detection limit, water solubility, fluorescence pH invariance, stability, distinctive fluorescence from protein chromophores, and ability to be detected using normal and reversed phase chromatography techniques.

We have incorporated these methods and reagents into our Marker Gene^TM Carbohydrate Analysis/Detection Kit (M0272). This kit is also useful in other carbohydrate analyses including HPLC detection and purification, carbohydrate receptor analyses, enzyme inhibition studies, as well as detection of abberant glycosylation or disease. The kit is capable of quickly estimating and/or comparing the composition of the carbohydrates in such samples. Additional information on these techniques is available. Custom couplings and determinations can also be arranged if desired. Please contact our technical services department for further information. For more information about these assays and kits, please see the references below or visit our website.

• Naleway JJ, Cook GM, Coleman DJ (2008) " Analysis of Tissue-Specific and Inhibitor Directed Plant Glycan Composition by Fluorescent Labeling with 1,5-EDANS and Combined Gel-Electrophoretic, High-Performance Liquid and Thin-Layer Chromatography Techniques." Abstract B-P023, XXIVth International Carbohydrate Symposium, Oslo, Norway, July, 2008.
• Gao N, Lehrman M, (2003) “Alternative sources of reagents and supplies for fluorophore-assisted carbohydrate electrophoresis (FACE).”  Glycobiology 13(1): 1G -3G.
• Starr CM, Masada RI, Hague C, Skop E, Kolck JC, (1996) “Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates.” Journal of Chromatopgraphy A(720): 295-321.
• Hase S, Ibuki T, Ikenaka T, (1983) “Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins.” J. Biochem. 95: 197-203.
• Harvey DJ, Wing DR, Kuster B, Wilson IBH, (2000) “Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins.” J Am Soc Mass Spectrom 11: 564-571.
• Fu D, O’Neill RA, (1995) “Monosaccharide composition analysis of oligosaccharides and glycoproteins by high-performance liquid chromatography.” Analytical Biochemistry 227: 377-384.
• Wu W, Hamase K, Kiguchi M, Yamamoto K, Zaitsu K, (2000) “Reversed-phase HPLC of monosaccharides in glycoproteins derivitized with aminopyrazine with fluorescence detection.” Analytical Sciences 16: 919-922.

a-Mannosidase Assays for Golgi Mannosidase II Analysis

Post-translational modification of nascent proteins after biosynthesis involves a highly complex series of enzymatic reactions in the endoplasmic reticulum and Golgi apparatus of all cells. Biosynthesis of Asn-linked oligosaccharides on cellular and secreted glycoproteins is highly compartmentalized in eukaryotic cells, occurring in the membranes of the secretory pathway including the endoplasmic reticulum (ER), the Golgi complex, trans-Golgi network, and the transport vesicles between these compartments. First, a polyisoprenoid lipid-linked precursor oligosaccharide is transferred to Asn groups of the protein through the action of an oligosaccharide transferase complex to generate an amide linked oligosaccharide on Asn residues in Asn–X–Ser/Thr sequences of the protein in the ER. Then the N-glycans are trimmed by the excision of all three glucose residues as well as varying degrees of mannose removal. Oligosaccharide processing in vertebrate organisms results in the initial removal of four of the original nine mannose units to produce a Man5GlcNAc2 core. Following the addition of a single GlcNAc residue, two more mannose residues are removed in stages in the Golgi by a-mannosidase II to result in a GlcNAcMan3GlcNAc2 core. The final stage of oligosaccharide glycoprotein synthesis involves the branching and extension of the resulting oligosaccharides by Golgi glycosyltransferases. These elaborations provide for the great diversity of oligosaccharide structures that are attached to proteins in different cells and tissue types.

Recent interest in these processing enzymes in the ER and Golgi has been initiated to screen for treatment options in a variety of genetic enzymatic defects in processing enzymes leading to phenotypic lysosomal storage diseases such as a-mannosidosis. In particular, the human deficiency in Golgi a-mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Marker Gene introduced a new method to monitor intracellular mannosidase activity using a continuous assay format at the recent International Carbohydrate Symposium in Oslo, Norway, using the red fluorogenic substrate Resorufin a-D-mannoside (M1340). This method was evaluated for monitoring activity versus cloned human Golgi a-Mannosidase II as well as human lysosomal a-mannosidase.

This new substrate produces the red fluorophore Resorufin (M0202) that can be monitored using EX 550 nm and EM at 595 nm. Because the pKa of resorufin is 5.8, it allows continuous measurement of fluorescence turnover at or near physiological pH values found in acidic organelles such as lysosomes or the Golgi.  The Golgi Mannosidase II enzyme operates at a pH optimum of pH 5.75, while the lysosomal mannosidase enzyme has a pH optimum of 4.5. Our results indicate that this substrate is very useful in measuring Golgi a-Man II activity. Resorufin a-D-mannoside (M1340) also exhibited greater activity towards Golgi Mannosidase II  versus human lysosomal mannosidase, indicating it may be suitable for specific intracellular Golgi staining and activity analysis. Use of this new substrate in screening new therapeutics for a-mannosidosis or for use in inhibiting transport and secretion of peptides and proteins from cells is under development. For more information about this new substrate, please visit our website

• Naleway JJ, Coleman DJ, Cook GM, Kuntz D, Williamson SP, Sim L, Venkatesan M, Rose DR, (2008) "New Resorufin-Based Glycosidase Substrates for Use in Continuous Analysis of Glycosidase Activity in Acidic Organelles." presented at the XXIVth International Carbohydrate Symposium, Oslo, Norway, July, 2008.
• Coleman DJ, Studler MJ, Naleway JJ (2007) "A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-D-cellobioside." Anal. Biochem. 371: 146-153.
• Moreman KW, (2002) "Golgi a-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals." Biochim. Biophys. Acta 1573(3): 225-235.
• Li B, Kawatkar SP, George S, Strachan H, Woods RJ, Siriwardena A, Moremen KW, Boons GJ, (2004) "Inhibition of Golgi Mannosidase II with Mannostatin A Analogues: Synthesis, Biological Evaluation, and Structure-Activity Relationship Studies." ChemBioChem 5(9):1220-1227.

New Long-Wavelength Fluorophore Labels.

A new series of long-wavelength fluorescent probes have been developed by SETA BioMedicals, LLC. The new dyes are based upon squarine carbocyanine derivatives that exhibit extremely bright emission, have a low tendency for self-quenching even at higher D/P ratios, have high extinction coefficients in the 250,000 M–1cm–1 range and quantum yields as high as 70%. Compared with typical Cy3 and Cy5 dyes, the fluorescence lifetime in aqueous buffer solution is up to twice as long as that of Cy-dyes while the quantum yield is comparable therefore making them a perfect label for use in homogeneous fluorescence polarization assays. These new dyes, probes and labels cover the entire visible spectrum as well as the near infrared (NIR) and provide fluorescent tools for biomedical, pharmaceutical, and environmental research for applications in High-Throughput Screening (HTS) and Multiplexed Analysis Systems. Marker Gene Technologies, Inc. is currently working with SETA Biomedicals, LLC to bring you these probes. For more information about these new labels, please see the references below, or visit our website for more information.

• Bernhard Oswald, Leonid Patsenker, Josef Duschl, Henryk Szmacinski, Otto S. Wolfbeis, and Ewald Terpetschnig. Synthesis, Spectral Properties, and Detection Limits of Reactive Squaraine Dyes, a New Class of Diode Laser Compatible Fluorescent Protein Labels. Bioconjugate Chem., 10, 925-931 (1999).
• Bernhard Oswald, Frank Lehmann, Lydia Simon, Ewald Terpetschnig, and Otto S. Wolfbeis. Red Laser-Induced Fluorescence Energy Transfer in an Immunosystem. Analytical Biochemistry, 280, 272–277 (2000).
• Patsenker L., Kolosova O., Tatarets A., Fedyunyayeva I., Povrozin Ye., Yermolenko I., Kudryavtseva Yu., Terpetschnig E. Fluorescent Probes and Labels for Biomedical Applications. 10th Conference on Methods and Applications of Fluorescence (MAF-10), Salzburg (Austria), 9–12 September 2007, P. 154. Poster PRLS-36.

• 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.

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