Discovery of Neuro-regenerative Natural Products from Marine Algae and Cyanobacteria
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Transcript of Discovery of Neuro-regenerative Natural Products from Marine Algae and Cyanobacteria
Discovery of Neuro-regenerative Natural Products from Marine Algae and Cyanobacteria
Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick
Background
At present, there are an estimated 243,000 people living with spinal cord injuries in the United States
53% of new injuries occur within the 16-30 age group
Goals
Identify a small molecule from algae/cyanobacteria which exhibits activity in stimulating nerve regeneration
Many algae compounds are similar to retinoic acid and eicosanoids
A marine sponge compound, Lembehyne A, promotes neurite outgrowth
Known outgrowth promoters stop cell cycle progression, as do many of the lab’s characterized compounds
Neuro 2a Neuroblastoma Cells: A mouse cancer cell line deriving from neurons
Neuron: Cell with capability of transmitting electric signals, found in nervous system
Neurite: Long, branching outgrowth from a neuron
Differentiate: Cells mature, adopt distinctive functions, less likely to divide
http://cancerweb.ncl.ac.uk
First – Defining Some Terms
Neuro 2a Cells with Neurites
Experimental Protocol
Neuro 2a cells are cultured in serum supplemented medium
Cells then exposed to novel marine extracts, observed in 24 hr increments
Neurite outgrowth compared against untreated control cells and ones treated with Lactacystin and with 8-Br-cAMP, known outgrowth promoters
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RPMI w/30μL DMSO5 μMLactacystin
Neurite Outgrowth Controls
Treated
Control
Choosing a Concentration
Concentration (μg/ml)
30 10 3 1 0.3 0.1F 91 74 19G 95 90 64H 97 96 95 66 21 11I 97 96 96 61 2114
54 F
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Values are % lethality
52 pure compounds
27 crude fractions
One worth mentioning…..
Symploca 1454 Fractions
1454 A 30 μg/mL: 0.53% 10 μg/mL: 0.27%
1454 B 30 μg/mL: .32% 10 μg/mL: 1.2%
1454 C 30 μg/mL: Toxic 10 μg/mL: .35%
Cont’d
1454 D 1 μg/mL: 0.55% 0.3 μg/mL: 1.5%
1454 E 10 μg/mL: 1.9% 3 μg/mL: .88%
1454 F 3 μg/mL: 4.0% 1 μg/mL: .48%
Cont’d
1454 G 1 μg/mL: 3.1% 0.3 μg/mL: 4.0%
1454 H 0.3 μg/mL: 5.2% 0.1 μg/mL: 0.9%
1454 I 0.3 μg/mL: 3.2% 0.1 μg/mL: 1.5%
Sub fractions
H-1 0.003 μg/mL: 4.5% 0.001 μg/mL: 0.8%
H-2 0.1 μg/mL: 1.1% 0.03 μg/mL: 1.0%
AU
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Minutes10.00 20.00 30.00 40.00 50.00 60.00 70.00
996 at 340.00996 at 216.00996 at 230.00996 at 254.00
Baseline 1
H-1 Peak
Baseline 2
Assay Results
H-1 Peak 0.03 μg/mL: 1.0% 0.01 μg/mL: 1.6%
Baseline 1 0.003 μg/mL: 1.8% 0.001 μg/mL: 0.5%
Baseline 2 0.003 μg/mL: 2.0% 0.001 μg/mL: 0.6%
Possible Synergisms?
0.03 μg/mL0.03 μg/mL
0.003 μg/mL
0.003 μg/mL
…..perhaps….
H-1 Peak and Baseline 1 2.3%
H-1 Peak and Baseline 2 4.3% 6.5% 4.3%
…..results, conclusions, and future plans…
Further investigation of Symploca compounds
Continued screening of crude fractions and newly purified compounds
Acknowledgements
Rebecca Medina
Gerwick Lab
Kevin Ahern & HHMI
References
Blanco et al., Cell Biol. Int. 2001, 25, 909
Fenteany et al., J. Biol. Chem.1998, 273, 8545
Aoki et al., Biochem. Biophys. Res. Com. 2001, 289, 558