The Effect of Centrophenoxine on Parkinson’s Disease Victoria Wei
Need
Taken from Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278-282.
Figure 1 The amount of Parkinson’s disease cases per 100,000 people in the United States as age increases
Knowledge Base
Parkinson’s disease is a brain disorder involving the nerves.
http://www.spinstudios.co.uk/sa/pa3.jpg
Figure 2 The effects of Parkinson’s disease
Knowledge Base
Figure 3 The life cycle of C. eleganshttp://www.wormatlas.org/handbook/fig.s/IntroFIG6.jpg
Knowledge BaseLipofuscin is an auto-
fluorescent age pigment which is found in people with neurodegenerative diseases. (Gray, et. al., 2005)
Figure 4 Lipofuscin in neurons of the human brain. http://www.innovitaresearch.org/news/res/06042501_01.jpg
Knowledge BaseCentrophenoxine is an
anti-aging medicine which slows the accumulation of lipofuscin.
Schneider, et. al. (1977)http://commons.wikimedia.org/wiki/File:Centrophenoxine.svg
Figure 5 Molecular structure of centrophenoxine
Literature ReviewCaldwin, et. al. (2008)
Figure 7 Both images taken from Caldwin, Guy A.; K.A. Caldwell. “Traversing a wormhole to Combat Parkinson’s disease.” Disease Models and Mechanisms. Volume 1. pp.000-000. 2008.
Figure 6
Literature ReviewSutphin, et. al. (2009)
Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.
Figure 8Auto fluorescent pigments present in Day 4 and Day 8 C. elegans
Literature ReviewGerstbrein, et. al.
(2008)
Figure 9 Auto fluorescent pigments present in Day 4 and Day 8 C. elegans.
Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans.
Literature ReviewApplication of centrophenoxine to the C. elegans
decreased the rate of lipofuscin accumulation by an average of 41.3%. (Shulkin, et. al., 1978)
Figure 10 Table displaying effect of centrophenoxine on lipofuscin in C. elegans
Shulkin, D.J.; B.M. Zuckerman. “Spectrofluorometric analysis of the effect of centrophenoxine on lipofuscin accumulation in the nematode C. elegans.” Age. Volume 5. Pp. 50-53. 1982.
PurposeThe purpose of the experiment is to observe the effects
of centrophenoxine on Parkinson’s disease in C. elegans
HypothesisNull- the symptoms of Parkinson’s disease will remain the same with
or without the application of centrophenoxine.Alternate- the symptoms of Parkinson’s disease will lessen with the
application of centrophenoxine.
The Effects of Centrophenoxine on the development of Parkinson’s disease in C. elegans
Wild type C. elegans and ham-1 (ot339) C. elegans obtained from the Caenorhabditis Genetics Center
N=80
Wild type C. elegans: N=40 ham-1(ot339) C. elegans: N=40
6.8 mM centrophenoxine will be applied to the Nematode Growth Media for 21 days.
Use of 4',6-diamidino-2-phenylindole (DAPI) to observe the amount of auto fluorescent pigment- lipofuscin- in C. elegans. GFP filters may also be used to observe whether the C. elegans DA neurons will be affected by the application of centrophenoxine
Statistical analysis
Given 6.8 mM centrophenoxineN=20
Control N=20
Given 6.8 mM centrophenoxineN=20
Control N=20
ProtocolC. elegans are grown
in petri dishes containing Nematode Growth Media (NGM) from Carolina Biological and fed U.V. killed Escherichia coli.
Figure 11 Culturing the C. elegans in Petri dishes
Picture by author
ProtocolBoth Ampicillin and 5-Fluoro-2 -deoxyuridin will be ′
used with NGM in the petri dishes with C. elegans
http://upload.wikimedia.org/wikipedia/commons/b/b6/Ampicillin_structure.svg
http://www.sigmaaldrich.com/structureimages/30/mfcd00006530.gif
Figure 12 Ampicillin Figure 13 FUDR
E.coli + NGM with centrophenoxine + Ampicillin + FUDR using the DAPI and GFP filter to observe amount of lipofuscin and fluorescence present in both C. elegans groups
Protocol
Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.
Figure 14 Age synchronization of C. elegans
Protocol
Figure 15 4',6-diamidino-2-phenylindole (DAPI)
http://upload.wikimedia.org/wikipedia/commons/7/7a/DAPI.png
Figure 16 Fluorescence microscopehttp://www.bcgsc.ca/people/msleumer/htdocs/fact_sheet/image004.jpg
Figure 17 GFP expressed in C. elegans
BudgetVendor Cat# Item Qty. Unit $ Total $
Caenorhabditis Genetics Center GS1214 ham-1(ot339) C. elegans 1 $7 $7
Caenorhabditis Genetics Center AB1 Wild type C. elegans 1 $7 $7
Sigma D9542-5MG DAPI 1 $51.60 $51.60 Sigma S2002 Sodium azide 1 $21.20 $21.20
Sigma F0503-100MG FUDR 1 $117 $117
Sigma SLC5377-25G Centrophenoxine Hydrochloride 1 $97.82 $97.82
Carolina Biological 741270 Petri dishes 10 $6.45 $64.50
Carolina Biological 216880 Ampicillin dry powder 1 $43.25 $43
Carolina Biological 173520 Nematode Growth Agar 2 $6.25 $12.50
Carolina Biological OP50 E. coli 1 $7 $7
Invitrogen D21490 DAPI 1 $116.00 $116.00
Nova-tech 1482FLi Fluorescence Microscope 1 $3,760 $3,760
Tritech MINJ-F-EFB GFP filter set 1 $938.38 $938 Total cost $5,243
Do-abilityAvailable for Purchase:
ham-1(ot339) and wild type C. elegans strains from CGC DAPI and Sodium Azide from SigmaNGM and OP50 E.coli from Carolina BiologicalCentrophenoxine purchasable from Science Lab.com
Equipment already Acquired:DAPI filter, GFP filter, fluorescent microscope
Bibliography "About Parkinson Disease." National Parkinson Foundation. <”http://www.parkinson.org/Page.aspx?pid=225”>. 1996-2007. Braungart, Evelyn; Gerlach, Manfred; Riederer; Peter, Baumeister, Ralf; and Hoener, Marius C. “Caenorhabditis elegans MPP+ Model of
Parkinson’s Disease for High-throughout Drug Screening.” Neurodegenerative Disease. 2004. Volume 1: pgs 175-183. Caldwin, Guy A.; K.A. Caldwell. “Traversing a wormhole to Combat Parkinson’s disease.” Disease Models and Mechanisms. Volume 1. pp.000-
000. 2008. Colleta, Susan. Introduction to C. elegans. Waksman Student Scholars. <http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/
worms.html>. 2009 Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and
dietary restriction in Caenorhabditis elegans. Hall, D. H.; Z. F. Altun. “C. elegans Atlas.” Genetics Research, 90 , pp 375-376. 2008. Hunt, Sara S. The Aging Process. Washington D.C. April 2004. Kenyon, Cynthia. “Environmental Factors and Gene Activities That Influence Life Span” C. elegans II. Cold Spring Harbor Press. 1997. Kisiel, Marion J.; B. Zuckerman. “Effects of Centrophenoxine on the Nematode Caenorhabditis Briggsae” Age. Volume 1. Pp.17-20. January 1978. Mc Naught, KS; P. Jenner. “Proteasomal function is impaired in substantia nigra in Parkinson's disease “ Neuroscience Letters. Volume 297. pp.
191-194. 2001. O'Riordan ; A.M. Burnell. Intermediary metabolism in the dauer larva. II. The glyoxylate cycle and fatty acid oxidation. Comp. Biochem. Physiol.
Volume 95. pp. 125-130. 1990. Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278-
282. Schneider, Howard F.; C. Nandy. “Effects of Centrophenoxine on Lipofuscin Formation in Neuroblastoma Cells in Culture” Journal of Gerontology.
Volume 32. Pp. 132-139. 1997. Shulkin, D.J.; B.M. Zuckerman. “Spectrofluorometric analysis of the effect of centrophenoxine on lipofuscin accumulation in the nematode C.
elegans.” Age. Volume 5. Pp. 50-53. 1982. Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. “What is Parkinson’s?” American Parkinson Disease Association West Coast Office. <“http://www.apdawest.org/WhatIsParkinsons.html#2”>.
2009.