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Research Program
Schistosomes, or blood flukes, are flatworms that parasitize humans and cause schistosomiasis, a widespread tropical disease. Using molecular biological approaches, my laboratory is working to better understand the nervous system of schistosomes such as Schistosoma mansoni. The ultimate goal of this work is to provide possible molecular targets for new, potent and specific antiparasitic agents.
Current Projects
Calcium channels reside in cell membranes, open in response to a voltage change across the cell’s membrane, and allow calcium to flow into the cell. This calcium current has two consequences: first, it contributes to the propagation of electrical signals in excitable cells; second, it changes the level of calcium within the cell. Calcium is an important messenger molecule in biological systems and regulates a variety of cellular events, including muscle contraction, neurosecretion and changes in gene expression. Calcium channel ß subunits are proteins that are associated with the calcium channel subunit (α1) that actually forms the pore through which ions flow. The ß subunit modulates various properties of these α1 subunits.
Praziquantel is the drug of choice against schistosomiasis, yet the molecular mechanism by which it acts remains unclear. My lab has found that a particular component from schistosome calcium channels – namely, a certain type of ß subunit – confers praziquantel sensitivity to other calcium channels. This ß subunit is quite unique because it lacks specific sites at which a particular enzyme can add phosphate groups to the protein. We believe that those missing phosphorylation sites are critical determinants of its unusual functional properties and pharmacological sensitivities.
Low concentrations of praziquantel cause a rapid influx of calcium into the cells of the worm. Our results indicate that this is due to increased movement of calcium through calcium channels. This ability to confer praziquantel sensitivity is dependent upon the lack of the specific phosphorylation sites that are found in other ß subunits. If we artificially create those phosphorylation sites in the schistosome ß subunit, it no longer confers praziquantel sensitivity. If we remove those sites from other ß subunits that normally have them, those mutated ß subunits now confer praziquantel sensitivity. Based on these results, we are examining ß subunits in other praziquantel-sensitive organisms, as well as in strains of schistosomes that are resistant to the drug. We are also continuing to examine other properties of schistosome calcium channels, such as their levels of phosphorylation and the physical interaction between the α1 and ß subunits.
We continue our collaboration with Leonid Moroz at the Whitney Laboratory of the University of Florida to examine nitric oxide pathways in schistosomes. Nitric oxide is a gaseous intercellular signalling molecule that has been implicated in multiple physiological functions. These crucial roles for nitric oxide, as well as others we may yet discover, make the nitric oxide system a tempting potential target for new antiparasitic agents.
| Robert M. Greenberg, Ph.D. |
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Education:
1983, Ph. D. University of Virginia
1977, B.A. The Johns Hopkins University |
Selected Publications:
Kohn, A.B., Lea, J., Moroz, L.L, and Greenberg, R.M. Schistosoma mansoni: Use of a fluorescent indicator to detect nitric oxide and related species in living parasites Experimental Parasitology, in press.
Greenberg, R.M., Roberts-Misterly, J.M., and Anderson, P.A.V. The evolution of voltage-gated Na+ channels: were algal toxins involved? In press.
Greenberg, R.M. (2005) Are Ca2+ channels targets of praziquantel action? Int. J. Parasitol. 35, 1-9.
Greenberg, R.M. Ca2+ signaling, voltage-gated Ca2+ channels, and praziquantel in flatworm neuromusculature. Parasitology 131, S1-S12.
Greenberg, R.M. Praziquantel: Mechanism of action. In: "Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Control" (A. Maule, ed.). CABI. Invited review, in press.
Kohn, A.B., J.M. Roberts-Misterly, P.A.V. Anderson, and R.M. Greenberg. 2003. Creation by mutagenesis of a mammalian calcium channel beta subunit that confers praziquantel sensitivity to a mammalian calcium channel. Int. J. Parasitol. 33: 1303-1308.
Kohn, A.B., J.M. Roberts-Misterly, P.A.V. Anderson, N. Khan, and R.M. Greenberg. 2003. Specific residues in the Beta Interaction Domain of a schistosome calcium channel beta subunit are key to its role in sensitivity to the antischistosomal drug praziquantel. Parasitology 127: 349-356.
Kohn, A., Anderson, P., Roberts-Misterly, J. and Greenberg, R. (2002) Role of schistosome calcium channel subunits in praziquantel action. In: Proceedings of the 10th International Congress of Parasitology - ICOPA X. (Monduzzi, Ed.) Bologna, pp. 121-126.
Kohn, A. B., Anderson, P.A.V., Roberts-Misterly, J.M., and Greenberg, R. M. (2001) Schistosome calcium channel beta subunits: unusual modulatory properties and potential role in the action of the antischistosomal drug praziquantel. J. Biol. Chem. 276, 36873-36876.
Kohn, A.B., Lea, J.M., Roberts-Misterly, J.M., Anderson, P.A.V., and Greenberg, R.M. (2001) Structure of three high voltage-activated calcium channel alpha-1 subunits from Schistosoma mansoni. Parasitology 123: 489-497.
Anderson, P.A.V. and Greenberg, R.M. Phylogeny of ion channels. (2001). Comp. Biochem. Physiol. 129(1): 17-28.
Kohn, A.B., Moroz, L.L., Lea, J.M., and Greenberg, R.M. (2001) Distribution of nitric oxide synthase immunoreactivity in the nervous system and peripheral tissues of Schistosoma mansoni. Parasitology. 122 Pt 1: 87-92.
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