1. Mitochondrial Markers for VACS
Mariana Gerschenson, Ph.D., Director
Molecular Medicine and Infect. Diseases Laboratory
Hawaii AIDS Clinical Research Program
Departments of Medicine and Cell and Molecular Biology

2. Mitochondrial Medicine Laboratory Capabilities
Morphological, Biochemical, and Genetic Mitochondrial and Nuclear Studies
Research being done on: Human cells and tissues

3. Mitochondrial Assays Performed by MMID
Isolating total DNA and RNA
Real time PCR of mtDNA copies/cell, OXPHOS genes, apoptosis, adipocyte differentiation, etc.
Subcellular isolation and localization studies
Assessing mitochondrial function and integrity
Mitochondrial protein levels and enzyme activities (OXPHOS, AST, fatty acid synthesis)
Mitochondrial and cellular oxidative stress and antioxidants
Transmission electron microscopy
SNP analysis and microarray
Proteomics

4. Proteolytic Processing
HIV
ART
Cytokines
Multi-Hit Effects of
HIV, ART, and Cytokines
On Mitochondria
DNA polymerase-g
Apoptosis
Uncoupling
Phosphorylation
Transport
Proteolytic Processing
Oxidative Stress
Glycosylation
Gerschenson, M. and Brinkman, K. Mitochondrion, in press, 2004.

7. Mitochondrial DNA

8. Mitochondrial Markers for VACs
Measure mitochondrial AST and cytoplasmic AST in sera from patients
Measure systemic mitochondrial damage in PBMCs from patients by measuring mtDNA and mitochondrial 8-hydroxy-2’-deoxyguanosine

9. Analysis of mtDNA copies/cell by a real-time PCR
The upper panels show the slope of the standard serial dilutions for mtDNA
amplified in real time and their fluorescence traces. The lower panel is the
fluorescence trace of three experimental samples followed by their melt curve
analysis.

10. MtDNA Levels are Decreased in Cord Blood PBMCs of HIV (+) Pregnant Women treated with NRTIs
144 ± 101
N = 6
865 ± 331
N = 4
p=0.0026
Shiramizu, B. et al., JAIDS, 32; 370, 2003

11. Mitochondrial Gene Specific Repair Assay
Lane:
1. Control
2. +Fpg
3. Damaged -Fpg
4. Damaged +Fpg
Anson, R.M. and Bohr, V.A.,
Methods of Mol. Biol., 113:257, 1999
Lane

12. Pathway for the synthesis of aspartate family amino acids
Pathway for the synthesis of aspartate family amino acids. AAT=aspartate aminotransferase; AS=asparagine synthetase; AK=aspartate kinase; DS=dihydrodipicolinic acid synthase; HSDH=homoserine dehydrogenase. CS=cystathionine gamma synthase.

13. Measuring Mitochondrial AST in Sera
Immunoprecipitate
Antibody to
Cytoplasmic AST
Sera
Sera with
Mito AST

14. AST Enzyme Assay
Aspartate aminotransferase may be assayed spectrophotometrically in a coupled reaction with malate dehydrogenase in the presence of NADH at 340 nm at 25oC for 3-4 minutes.
Reagents
L-aspartate mM
2-oxoglutarate mM
NADH mM
Lactate dehydrogenase 5 u/ml
Malate dehydrogenase u/ml
Sodium phosphate buffer, pH mM
Worthington Biochemicals and Diagnostic Chemicals Limited
and

15. Human Mitochondrial and Cytoplasmic AST Antibodies Recognized AST in Human Sera
MITO CYTO MW
(kDa)
Lane:
1. MW marker
2. Porcine AST
3. Sera
4. MW marker
5. Porcine AST
6. Sera
7. Sera
120
100 80 60 50 40 30 20
AST
Lane

16. VACS Samples to be Analyzed

17. Leahi Hospital at the University of Hawaii

18. Molecular Medicine and Infectious Disease Laboratory
Photo:
Bruce Shiramizu, M.D.
Duy Trans
Brain Seavy
Daniel LiButti
Mariana Gerschenson, Ph.D.
Cecilia Shikuma, M.D.
Not in photo:
Lori Kamemoto, M.D.
Larry Day, M.D.
Franchette Pasqual
Jennifer Lloyd
Kimber Cochrane