Transcript What is Inflammatory Bowel Disease

Investigating Intestinal Alkaline Phosphatase
as a Risk Factor for Inflammatory Bowel
Disease
Vanessa Danquah
Guillemin Lab
What is Inflammatory Bowel
Disease ?
Intestinal Alkaline Phosphatase
Detoxifies Lipopolysaccharide in
Zebrafish

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IAP is an enzyme that interacts with the microbiota in the
gut.
It was proven that decreased IAP activity caused
inflammation in zebrafish intestine.
Is there a correlation between IAP
and Inflammatory Bowel Disease ?

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Is the function of
intestinal alkaline
phosphatase in mediating
bacterial-host
interactions
evolutionarily conserved?
Do polymorphisms in
human IAP alter
enzymatic activity and
contribute to IBD?
Measuring activity of IAP in
Drosophila gut
ng Pi/min/mg tissue
Wild Type Gut vs. Carcass
Wild Type Gut
Wild Type Carcass
Gender and Age Experiments
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Gender
Age
Day 0 -5 Gut Activity
0.35
0.30
0.30
0.25
0.25
0.20
0.15
0.10
0.05
0.00
Female Gut
Male Gut
ng Pi/min/mg tissue
ng Pi/min/mg tissue
Female Gut vs. Male
Day 0
0.20
Day 1
Day 2
0.15
Day 3
Day4
0.10
0.05
0.00
Day 5
L-Phenylalanine-IAP Inhibitor
L-Phenylalanine Inhibitor
Threshold
L-Phenylalanine Inhibitor
0.35
0.35
0.25
0.2
Control-Gut
Control-Carcass
L-phenyl 50 mM - Gut
0.15
0.1
0.05
0
L-phenyl 50 mM-Carcass
ng Pi/min/mg tissue
ng Pi/min/mg tissue
0.3
0.3
0.25
0.2
Control
10uM
20uM
30uM
0.15
40uM
50um
0.1
0.05
0
Searching for the gene or genes that
encode IAP in Drosophila
Female
X
2L
2R
3L
3R
4
X
2L
2R
3L
3R
4

Each deficiency stock deletes approximately
50 genes, one of which is the candidate for
encoding IAP.
Two chromosomal regions encode
IAP genes
Deficiencies 1-4
0.4
ng Pi/min/mg tissue
0.35
0.3
Wt
0.25
0.2
0.15
0.1
0.05
0
Df1
Df2
Df3
Df4
Human IAP Polymorphisms
R125H
P65L
H279L
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H279L
Different people have different amino acids within their IAP proteins at certain
locations

We will investigate whether these amino acid changes alter the enzyme’s activity
How did we create known IAP
variants?
Make IAP variants via site directed
mutagenesis
Clone IAP variants and wild type
construct into an expression vector
Transfect and express them in Chinese
Hamster Ovary Cells (CHO )
Assay for activity with pNPP substrate
Initial results of human IAP enzyme
activity in vitro
L-Phenylalanine Activity of
Transfected Cells
24
48
3:2
4:2
5:2
6:2
1
7:2
8:2 UT
Ratio of Transfection Reagent: DNA
Absorbance at 405 nm
Absorbance at 405nm
IAP Activity- Post Cell Transfection
Untreated
50mM L-phenyl
1
Conclusion and Future Directions
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Drosophila intestines have
high alkaline phosphatase
activity that is inhibited by
the mammalian specific IAP
inhibitor L-Phenylalanine
We have identified two
chromosomal regions in the
Drosophila genome that are
likely to encode Drosophila
IAP genes
We have established a cell
culture system to study
human IAP polymorphisms
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Further characterize the
genes within chromosomal
deletions that reduce IAP
activity
Continue to test IAP variants
and whether they alter
activity in vitro
Test whether expression of
human IAP variants in
Drosophila alter bacterialhost interaction in flies
Acknowledgements
Guillemin Lab
Dr. Karen Guillemin
Ms. Anica Wandler
Dr. Peter O’Day
SPUR 2009