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What did one cell say to the other? Laura Ruth
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hat are the wireless connections for cellular communication? What is the best way to eavesdrop, monitor, record, and decipher cell signaling conversations? To answer these questions, analytical chemists tap into intra- and intercellular signaling communication pathways using a variety of classical and new techniques, such as voltammetry, capillary electrophoresis-laser-induced fluorescence (CE-LIF), matrix-assisted laser desorption/ionization (MALDI) MS, and micellar electrokinetic chromatography (MEKC). Yet there are still plenty of opportunities to explore neural cell communication pathways. So how does a newcomer, who may not know a cytokine from a G-protein, begin?
Getting started Despite the huge number of Web sites devoted to cell signaling (see the boxes on pages 160 and 161), several researchers in the field say that old-fashioned books and articles are still a great place to start (1–5). Once you have the basic information about cell signaling and analytical chemistry, you’ll be ready to go to the Web to explore a few links. Read the definitions of a few key signal transduction terms—reception, transduction, and induction—and look at a diagram of a signal transduction pathway in plant cells on the 21st Century Biology page, which is aimed at pre-college students. Use the Terre Haute Center for Medical Education’s signal transduction page for a few basic definitions about receptors, tyrosine kinases, phospholipids, and other signal transducers. With your
eyes and ears tuned to cell signaling, it is easier to browse the signal transduction section of the WWW Virtual Library of Cell Biology. This site provides a gateway to considerable information about a wide range of cell signaling topics, such as calcium binding protein structures and pathways at the EF-Hand Calcium-Binding Proteins Data Library; a map of mam-
malian mitogen-activated protein (MAP) kinase pathways; synaptic transmission (part of the Multimedia Neuroscience Education Project); a signal transduction mailserver run by the Melbourne signal transduction group; and a Bionet.cellbiol newsgroup for insulin researchers. The links at Columbia University, Demokritos (the National Centre for
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Getting started
Mammalian MAPK Pathways Map http://kinase.oci.utoronto.ca/pages/maps.html
21st Century Biology http://www.sidwell.edu/~bio21/ plants/back/signal.html
Melbourne Signal Transduction Group http://grimwade.biochem.unimelb.edu.au/ sigtrans.html
Bionet.cellbiol.insulin newsgroup http://www.bio.net/hypermail/INSULIN-ACTION
Multimedia Neuroscience Education Project http://www.williams.edu/imput/
Chemistry of Drugs and the Brain Seminar http://www.emory.edu/CHEMISTRY/ justice/chem190j/relatedNTs.htm
Ohio University College of Osteopathic Medicine http://lr.oucom.ohiou.edu/cvphysiology/ BP011.htm
Columbia University http://www.columbia.edu/cu/biology/ courses/c2006/links/signallinks.html COPE: Cytokines Online Pathfinder Encyclopaedia http://www.copewithcytokines.de Demokritos http://www.demokritos.gr/ib/group1.html EF-Hand Calcium-Binding Proteins Data Library http://structbio.vanderbilt.edu/cabp_database Ethylene in Signal Transduction http://www2.kenyon.edu/depts/biology/ edwards/project/wendy/ecker.htm Fred Hutchinson Cancer Research Center http://www.fhcrc.org/education/courses/ cancer_course/basic/signal.html * Available only to subscribers.
Scientific Research in Greece), and Ohio University College of Osteopathic Medicine are all useful resources for learning more about a few common signaling molecules and pathways, such as G-proteins, G-protein-coupled receptors, and cAMP. Among the links on the Columbia University page are several animations, two of which—“Production of IP3 and Ca2+ Ion Wave” and “Endocrine System”—are particularly educational and entertaining. Details about more specialized cell signaling systems are also available. Click on Emory University’s online seminar, “Chemistry of Drugs and the Brain”, for information about neurotransmitters. Find an introduction to the Rel/NF-B signal transduction pathway at the Boston University site. Pick up cancer signal transduction at the Fred Hutchinson Cancer Research Center page. Curious about the role of an ethylene molecule and plant signal transduction? Check out one of the Kenyon College Web pages. The basic information about various cell signaling pathways and molecules prepares you to explore a variety of sig160 A
Rel/NF-B B Signal Transduction Pathway http://people.bu.edu/gilmore/nf-kb/ mainfrpy.html Signal Transduction Knowledge Environment* http://www.stke.org/ Signaling Pathway Database http://www.grt.kyushu-u.ac.jp/spad/ menu.html Terre Haute Center for Medical Education http://web.indstate.edu/thcme/mwking/ signal-transduction.html Transpath Signal Transduction Browser http://transpath.gbf.de WWW Virtual Library of Cell Biology http://vl.bwh.harvard.edu/ signal_transduction.shtml
nal transduction molecule databases. Search the Signaling Pathway Database’s catalog of extracellular signal molecules for integrated genetic and signal transduction system information. Learn about cytokines as signal transducers with the Cytokines Online Pathfinder Encyclopaedia, also known as COPE. Last but not least, investigate a variety of signal transduction molecules in the Transpath Signal Transduction Browser. One of the most ambitious sites is Science’s Signal Transduction Knowledge Environment. Among the site’s many features are “This Week in Signal Transduction”, which highlights several research papers each week, and “Perspectives”, which allows authors to give their viewpoints or opinions. The fledgling “Connections Map”, provides graphical representations and textual summaries of selected pathways. And the “Virtual Journal” gives users access to full-text manuscripts from dozens of journals.
Analytical techniques Fast cyclic voltammetry. Although analytical chemists have been using electro-
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chemical techniques since the 1950s, biologists have only been tuning into the uses of analytical chemistry since the 1970s. To learn about the history, development, and uses of voltammetry, check out the primer, “In Vivo Detection of Neurotransmitters with Fast Cyclic Voltammetry”, at the Queen Mary and Westfield College neuroscience page. Also see Adrian Michael’s site at the Chevron Science Center at the University of Pittsburgh. Michael develops microelectrodes and microsensors to monitor neurotransmitters, such as acetylcholine, which seem to play a role in Alzheimer’s disease. CE-LIF. Because CE-LIF can separate and detect tiny amounts of material, it is well suited to studies of neurotransmitters. Among the researchers using CE-LIF this way is Norm Dovichi at the University of Washington–Seattle, whose lab developed a technique to derivatize primary amine-containing neurotransmitters with the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde, or FQ. Basic information about CE, LIF, and several possible applications is available at Dovichi’s Web site. More extensive information about CE
Analytical techniques and outlook Analytical Chemistry Springboard, Umeå University http://www.anachem.umu.se/jumpstation.htm CE and CEC http://www.ceandcec.com Environmental Engineering Lab http://www-ec.njit.edu/~hsieh/ ene669/ce.html Molecular Probes Web Handbook http://www.probes.com/handbook/ sections/1801.html Multimedia Shopping for a Mass Spec* http://pubs.acs.org/isubscribe/journals/ ancham-a/72/i05/html/webworks.html NIGMS Glue Grant http://www.nigms.nih.gov/funding/ gluegrant_release.html Queen Mary and Westfield College Neuroscience http://www.qmw.ac.uk/~physiol/ aboutFCV.html * Available only to subscribers.
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can be found at the CEandCEC.com site. For information about some of the fluorescent probes that can be used to visualize Ca+2 regulation, receptors, and other signal transducers, see chapter 18.1 of the Molecular Probes Web Handbook. MEKC and CZE. For a detailed explanation of CE, MEKC, and CZE, see the Environmental Engineering Lab page at the New Jersey Institute of Technology. You can monitor a variety of molecules in vivo, including primary amines, by linking MEKC and CZE to microdialysis and LIF. For example, Bob Kennedy’s laboratory at the University of Florida has consistently resolved glutamate and aspartate using CZE-LIF (6). Amazingly, with MEKCLIF, they have separated 11–20 different amino acids. The researchers have also directly compared the performance of CZE- and MEKC-LIF. MS. The MS resource page of the Analytical Chemistry Springboard at Umeå University (Sweden) is a good place to start looking for information on MS. Analytical Chemistry subscribers can also access Kermit Murray’s earlier WebWorks column, “Multimedia Shopping for a Mass Spec”, for introductory as well as specialized information, electronic mailing lists, and relevant usenet groups. MALDI time-of-flight MS is good for characterizing peptides in tissues and cells, and Jonathan Sweedler’s lab at the University of Illinois develops MALDI techniques to characterize neuropeptides from single cells and biological tissues (7–9). To further expand MALDI’s biological uses, Jim Jorgenson’s laboratory at the University of North Carolina–Chapel Hill has combined it with microcolumn LC (10). Fluorescence microscopy. Edward Yeung at Iowa State University and Ames Laboratory–U.S. Department of Energy demonstrates how native fluorescence microscopy complements other analytical chemistry techniques, such as MS, microelectrodes, LC, CE, NMR, and vibrational spectroscopy (11). His lab has also compared bio/chemiluminescence, UV absorption, and electrochemical methods for measuring intracellular ATP (12).
Cell signaling frontiers
Researchers’ Web sites
Subcellular analysis. Andrew Ewing at Pennsylvania State University thinks subcellular analysis is one of the next frontiers of analytical chemistry and cell signaling research. Ewing develops capillary and chip electrophoresis (with electrochemical, laser-based, and mass spectrometric detection), electrochemistry (e.g., ultra-small electrodes, carbon electrodes, and nanometer electrodes), and molecular imaging (e.g., MALDI and secondary ion MS) methods to investigate exocytosis, neurotransmitter release and uptake, and gene expression. Future tools. Applying analytical tools, while remaining cognizant of the body of knowledge in neurobiology, is the way to investigate cell-to-cell signaling in the brain, according to Mark Wightman at the University of North Carolina–Chapel Hill. Wightman develops microsensors and microelectrodebased techniques capable of detecting zeptomolar concentrations of analytes. He says that good electrochemical methods for studying the neurotransmitters known as catecholamines currently exist, and in the future, techniques such as multidimensional NMR, MS, or Raman or fluorescence microscopy may visualize fluctuations in chemical substances in real time and provide additional information. However, he cautions that these methods are not yet mature enough to determine what their contributions will be.
Norm Dovichi, University of Washington–Seattle http://depts.washington.edu/chemfac/ dovichi.html Andrew Ewing, Pennsylvania State University http://neuron.chem.psu.edu/ Jim Jorgenson, University of North Carolina–Chapel Hill http://www.unc.edu/depts/chemistry/ faculty/jwj/cfjwj01.html Bob Kennedy, University of Florida http://www.chem.ufl.edu/kennedy.html Adrian Michael, University of Pittsburgh http://www.chem.pitt.edu/faculty/ michael.html Jonathan Sweedler, University of Illinois http://www.beckman.uiuc.edu/faculty/ sweedler.html Mark Wightman, University of North Carolina–Chapel Hill http://www.unc.edu/depts/rmwgroup Edward Yeung, Ames Laboratory– U.S. Department of Energy http://www.fi.ameslab.gov/PIINFO/yeung.htm
Laura Ruth is a freelance writer who has written for a variety of publications in the areas of business, education, health, science, technology, and market research.
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Outlook Because cell-to-cell signaling is intrinsic to so many different systems, there is a demand for old and new technology development to investigate a huge number of scientific questions. Recognizing the interdisciplinary nature of cell signaling research, the National Institute of General Medicine Sciences created a “glue grant”. The grant forms a consortium of ~50 scientists around the country who will investigate B-cells and cardiomyocytes. Many analytical chemistry techniques will play a key role alongside proteomics, genomics, informatics, and other scientific disciplines in exploring cell-to-cell signaling and figuring out what one cell may say to another.
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Nicholls, J. G.; Martin, A. R.; Wallace, B. G. From Neuron to Brain, 3rd ed., Sinauer Associates, Inc.: Sunderland, MA, 1992. Kandel, E. R.; Schwartz, J. H.; Jessell, T. M. Principles of Neural Science, McGraw-Hill: New York, 1999. Trends in Anal. Chem. 1995, 14 (4). Adams, R. N. Anal. Chem. 1976, 48, 1128–1138. Stamford, J. A.; Justice, J. B., Jr. Anal. Chem. 1996, 68, 359 A–363 A. Lada, M. W.; Kennedy, R. T. Anal. Chem. 2000, 68, 2790–2797. Li, L.; Garden, R. W.; Romanova, E. V.; Sweedler, J. V. Anal. Chem. 1999, 71, 5451–5458. Li, L., et al. Anal. Chem. 2000, 72, 3867–3874. Rubahkin, S. S.; Garden, R. W.; Fuller, R. R.; Sweedler, J. V. Nat. Biotechnol. 2000, 18, 172–175. Hsieh, S., et. al. Anal. Chem. 1998, 70, 1847–1852. Yeung, E. S. Anal. Chem. 1999, 71, 522 A–529 A. Wang, Z.; Haydon, P. G.; Yeung, E. S. Anal. Chem. 2000, 72, 2001–2007.
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