Fluorescent and Luminescent Tools for Life Science
   

News Archive - June, 2007 - Volume 7, Number 6

LacZ and neoR used to Identify Stem Cells Generated from Mouse Fibroblasts.

The ability to reprogram terminally differentiated cells into pluripotent embryonic stem (ES) cells has been accomplished by two teams of researchers including Prof. Shinya Yamanaka and coworkers at Kyoto University and Drs. Wernig, Jaenisch and Hochedlinger of Massachusetts General Hospital and the Harvard Stem Cell Institute.  The Kyoto group were able to identify a minimal set of factors required to induce the developmental potential of an ES cell into somatic cells.  Using previous evidence that ES cells have reprogramming capabilities, and forcing expression of sets of ES cell-specific transcription factor genes into  somatic cells, they have induced them to take on an embryonic character. The two groups of researchers reported generation of pluripotent stem cells from mouse adult fibroblasts by introducing a set of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions.  In order to identify the set of factors needed for reprogramming, mouse fibroblasts were retrovirally transformed using a drug selection cassette (b geo) that contained both lacZb-galactosidase and neomycin resistance genes under the control of a promoter active only in ES cells (Fbx15).  Both mouse embryonic fibroblasts and fibroblasts from the tail tips of adult mice (MEFs and TTFs, respectively) were subjected to this reprogramming strategy.  The authors deduced that nuclear reprogramming had taken place if these fibroblasts both expressed β-galactosidase activity and became resistant to high concentrations of neomycin.  After identifying the minimal set of transcription factors necessary to revert them to an embryonic state, the cells exhibited the morphology and growth properties of ES cells.  In addition, subcutaneous transplantation of new ES cells into nude mice resulted in tumors in a variety of tissues.  Following injection into blastocysts, the new ES cells were shown to contribute to normal mouse embryonic development.  These data demonstrated that pluripotent stem cells could be generated from fibroblast cultures by the addition of only a few defined factors.  Potentially, a similar methodology could be used to generate human ES like cells.  For more information about these techniques and methods, please visit our website or see the references below.

  • Takahashi, K., Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors.Cell 126: 663–676 (2006).
  • Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R. “In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007 Jun 6; [Epub ahead of print].
  • Cowan CA, Atienza J, Melton DA, Eggan K. 2005 “Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells.” Science. 309(5739):1369-73.
  • Rodolfa KT,Eggan K. (2006) “A transcriptional logic for nuclear reprogramming.Cell. 126(4):652-5.
  • Okita, K., Ichisaka, T. & Yamanaka, S. Nature doi:10.1038/nature05934 (2007).
  • Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R.,  Plath, K., Hochedlinger K., (2007) Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution Cell Stem Cell

a-(1-4)-di-Galacturonic Acid.

Degradation of polysaccharides, such as pectins in plants and glycosaminoglycans in animal tissue, into smaller oligosaccharides is a crucial process for many plant and animal pathogens.  Such pathogens include the plant pathogenic bacteria belonging to Erwinia and the enterobacterial species belonging to Yersinia.  While the many stages of polysaccharide degradation are well understood, further catabolism of the hydrolysis products within the bacterial cell are not well understood.  Recently, researchers at the University of Victoria, British Columbia have characterized a periplasmic binding protein in Yersinia enterocolitica known as TogBTogB has a vital role in transport of polysaccharide hydrolysis products across lipid bilayers into the cytoplasm, such as trigalacturonic acid, unsaturated digalacturonic acid, and saturated digalacturonic acid, also known as alpha-DGalU(1-4)DGalU (Product M0095).  TogB binds these oligosaccharides and delivers them to the TogMNA2transport protein embedded in the lipid bilayer, allowing transport into the cytoplasm.  The binding site of TogB has been characterized when unbound, as well as when bound to various oligosaccharides.   For more information about these assays and systems, please see our website or see the references below.

  • Abbot DW, Boraston AB.  (2007)  Specific Recognition of Saturated and 4,5-Unsaturated Hexuronate Sugars by a Periplasmic Binding Protein Involved in Pectin Catabolism.  J. Mol. Biol. 369: 759-770.
  • Gouvion C, Mazeau K, Heyraud A, Taravel FR, Tvaroska I. (1994) “Conformational study of digalacturonic acid and sodium digalacturonate in solution.>Carbohydr Res. 261(2):187-202.
  • San Francisco, MJD, Xiang, ZX, Keenan RW, (1996) “Digalacturonic Acid Uptake in Erwinia chrysanthemi.” 9(2): 144-147.

A New Zinc-Binding BODIPY Probe.

Ionic zinc plays a diverse role in cells, acting variously as enzymatic cofactor, structural element or second messenger.  It is an essential component of many enzymes and transcription factors (e.g., carbonic anhydrase, zinc finger proteins).  Neurons in the brain contain a relatively large pool of free Zn+2 stored in vesicles near the synapse.  Zn+2 serves to modulate the function of glutamate receptors and is released by excitatory signals.  In the pancreas, Zn+2 is secreted along with insulin by pancreatic b-cells.   Zn+2 is also known to suppresses apoptosis, and induce the formation of b-amyloid, which may be related to the etiology of Alzheimer’s disease. Although Zn+2 has many physiologically important roles, the mechanisms involved in its action are still poorly understood.  Therefore, several chemical tools for measuring Zn+2 in living cells have been developed.  Fluorescent probes based on quinoline, fluorescein, other fluorophores or proteins, have been reported, but lack specificity or pH insensitivity.

Recent work from the laboratory of Professor Tetsuo Nagano and co-workers at the University of Tokyo have introduced a new BODIPY-based derivative as a specific chelator of Zn+2 ion.  They prepared derivatives of 1,3,5,7-tetramethyl-8-phenyl-boron dipyrromethene (BODIPY) at the 8-position wherein complexation by zinc changed the electron density of the fluorophore, producing enhancement of fluorescence intensity. The new probe had advantages of less sensitivity to solvent polarity and pH than fluorescein-based probes and was not influenced by other cations, such as Na+ , K+, Ca+2, and Mg+2, which exist at high concentrations under physiological conditions.  These new reagents represent one of the first known BODIPY-based functional probes for use in live cells.   For more information about these new reagents, please see our website or the references below.

  • Koutaka, H., Kosuge, J., Fukasaku, N., Hirano, T., Kikuchi, K., Urano, Y., Kojima, H., Nagano, T., (2004)” A Novel Fluorescent Probe for Zinc Ion Based on Boron Dipyrromethene (BODIPY) Chromophore”  Chem. Pharm. Bull. 52(6): 700-703.
  • Maruyama S., Kikuchi K., Hirano T., Urano Y., Nagano T., (2002) A Novel, Cell-Permeable, Fluorescent Probe for Ratiometric Imaging of Zinc Ion. J. Am. Chem. Soc., 124: 10650—10651.
  • Hirano T., Kikuchi K., Urano Y., Nagano T., (2002) “Improvement and Biological Applications of Fluorescent Probes for Zinc, ZnAFs.” J. Am. Chem. Soc., 124: 6555—6562.
  • Hirano T., Kikuchi K., Urano Y., Higuchi T., Nagano T., (2000) “Highly Zinc-Selective Fluorescent Sensor Molecules Suitable for Biological Applications.” J. Am. Chem. Soc. 122: 12399—12400.
  • Kay AR, Tóth K., (2006) “Influence of Location of a Fluorescent Zinc Probe in Brain Slices on Its Response to Synaptic Activation J. Neurophysiol. 95:1949-1956.

New Thiol Reactive Probes.

Although several fluorescent or chemiluminescent reagents have been developed for measurement of intracellular thiols (Ellman’s Reagent: 5,5'-dithiobis(2-nitrobenzoate) (DTNB), fluorescein-5-maleimide, 5-(bromomethyl)fluorescein, tetramethylrhodamine-5-iodoacetamide, ABD-F, etc.) these reagents often require separate separation steps, or are only sparingly water-soluble.  Recently, work from the laboratory of Professor Hatsuo Maeda and co-workers at Osaka University have developed a series of elegant fluorescein derivatives that have a pendant 2,4-dinitrophenylsulfonyl ester group that, upon reaction with thiols, produce highly fluorescent fluorescein derivatives that can be easily measured.  In addition, these derivatives have also been adapted to measure several enzyme activities, including aceytlcholinesterate by using acetylthiocholine or butyrlthiocholine as substrates.  In addition, by adjusting the pH of the assay, these reagents have also been found useful to detect selenol groups, in which the sulfur in thiol compounds is substituted by selenium (Se).  This makes them suitable for measuring selenoproteins.  For more information about these new reagents, please visit our website or see the references below. 

  • Maeda, H., Katayama, K., Matsuno, H., Uno,T., (2006) “3’-(2,4-Dinitrobenzenesulfonyl)-2’,7’-dimethylfluorescein as a Fluorescent Probe for Selenols”  Ange. Chem. Int. Ed. 45(11): 1810 – 1813.
  • Hatsuo Maeda, Prof. *, Hiromi Matsuno, Mai Ushida, Kohei Katayama, Kanako Saeki, Norio Itoh (2005) “2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman's Reagent in Thiol-Quantification Enzyme Assays” Ange. Chem. Int. Ed. 44(19): 2922 – 2925.
  • Ellman, G.L. (1958) "A colorimetric method for determining low concentrations of mercaptans" Arch. Biochem. Biophys. 74, 443-450.
  • Toyo'oka, T., Imai K., (1984) “New Fluorogenic Reagent Having Halogenobenzofurazan Structure for Thiols: 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.” Anal. Chem. 56: 2461

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