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LIVING WITHOUT OXYGEN
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LITTORINA LITTOREA
Marine gastropod (periwinkle) Found on the
Atlantic coast Number of species decreases
drastically from south to north. Intertidal
zone highly variable environment
Intertidal zone
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INTERTIDAL ZONE
Ingonish Highlands, Cape Breton Island (Nova
Scotia, Canada)
Incredibly buff research scientist
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INTERTIDAL POOL

• Highly variable temperature, salinity, oxygen,
  pH
• Temperature oxygen fluctuate daily, seasonally
  and from pool to pool
• At low tide gill-breathers exposed to air causing
  hypoxia and/or anoxia
• At low tide in winter air temp gtgt colder than
  water tissue freezing

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Inside the Tide Pool
A temporary environment is created during
every LOW TIDE. Air exposure
Hypoxia
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METABOLIC RATE DEPRESSION
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QUANTIFYING METABOLIC RATE DEPRESSION

• Oxygen consumption
• Calorimetry
• ATP turnover rate
• Cross-over studies on pathway flux
• Enzyme kinetic /-P analysis
• Gene Regulation

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METABOLISM IN ANOXIA

• mRNA synthesis
• Protein synthesis
• Fuel use (incl. CHO)
• O2 consumed

ATP turnover to lt5 of normal
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In vitro Protein Synthesis
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a
P
A254
M
1 2 3 4 5 6 7 8 9 10
Polysome Analysis
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P
A254
M
Normoxia
1 2 3 4 5 6 7 8 9 10
P
Anoxia
A254
M
1 2 3 4 5 6 7 8 9 10
Recovery
P
A254
M
1 2 3 4 5 6 7 8 9 10
Polysome Analysis
30 Linear Sucrose Gradient 15
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PRINCIPLES OF METABOLIC ARREST

• 1. Metabolic rate reduction
• 2. Control by protein kinases(SAPKs, 2nd
  messenger PKs)
• 3. Selective gene activation

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PRINCIPLES OF METABOLIC ARREST

• 1. Metabolic rate reduction
• 2. Control by protein kinases(SAPKs, 2nd
  messenger PKs)
• 3. Selective gene activation

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ANOXIA INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Gene inactivation ( mRNA )
• Few Genes activated

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PROTEIN KINASES

• Covalent modification by phosphorylation
• Families of protein kinases PKA (cAMP),
  PKG (cGMP), CaMK (Ca2), PKC (Ca2, PL,DG)
• SAPKs daisy chain phosphorylations
• Regulation via interconversion of active vs
  subactive forms of protein substrates

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ENZYME CONTROL BY REVERSIBLE PHOSPHORYLATION
ATP
ADP
Protein Kinase
P
P
P
P
Protein Phosphatase
Pi
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Reversiblephosphorylation control of enzymes
P deP enzymesseparate on ionexchange columns
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• Energy production
• in low oxygen
• Glycolysis
• Pasteur effect Lactate Rate
• Metabolicdepression End product Rate
  alt

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REVERSIBLE PHOSPHORYLATION GLYCOLYSIS
P effect GP (-) HK -G6PDH
-PFK-1 - PFK-2 - PK -
Storey KB. 1996. Comp Biochem Physiol B 113, 23-35
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PROTEIN PHOSPHORYLATION GLYCOLYSIS

• Protein kinase A , cAMP
• PKG , cGMP
• Protein kinase C
• Protein phosphatase 1, 2A, 2C

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PATHWAY CONTROL IN MR DEPRESSION
Phospho / de-Phospho

• Glycolysis (GP, GS, PFK, PK)
• Fat synthesis (ATP-CL, ACC)
• CHO fuel use (PDH)
• Translation (eIF2a, eEF2)
• Ion pumps (NaK-ATPase, Ca-ATPase)

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ANOXIA INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Gene inactivation ( mRNA )
• Few Genes activated (1-2)

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ANOXIA INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Gene inactivation ( mRNA )
• Few Genes activated

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TURNING OFF GENESRole of Epigenetics
Epigenetics stable changes in gene
activity that do not involve changes in DNA
sequence. Common mechanisms - DNA
methylation - Histone modification/histone
variants - Regulatory non-coding RNAs
Hibernators! Frogs! Littorines ?
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Cuellar TL, McManus MT. J Endocrinol.
187(3)327-332, 2005.
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ANOXIA INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Gene inactivation ( mRNA)
• Few Genes activated

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ROLE OF TRANSCRIPTION

• Global rate of mRNA synthesis depressed.
  Method nuclear run-on
• Are selected genes up-regulated ?
• TO ASSESS GENE UPREGULATION
• What new mRNAs are created - cDNA
  library, Gene Chip

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GENE CHANGES IN Anoxic Littorina
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GENE CHANGES IN Anoxic Littorina
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ANTIOXIDANT ENZYMES
Willmore WG Storey KB - FEBS J 272, 3602-14
(2005) - Am J Physiol 273, R219-25 (1997).
- Mol Cell Biochem 170, 177-185 (1997)
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ANTIOXIDANT DEFENSE

• Iron storage - Ferritin (H L chains) -
  Transferrin receptor 2
• Antioxidant enzymes - SOD (1) - GST (M5, A2) -
  GPX (1, 4) - Peroxiredoxin 1

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Ferritin heavy chain

• Sequesters and stores iron
• Protein shell holds up to 4,500 atoms of iron
• Shell has 24 subunits in combining
  light (19 kDa) and heavy (21 kDa) subunits
• Iron sequestering reduces oxygen free radical
  production via the FENTON REACTION

70s radicals
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Ferritin
heavy chain
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FERRITIN
Protein
Polysome
- 18 kDa
C 24 h 72 h 1 h R
Monosome
a
a
Time course (hours)
Translation continues during anoxia
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FERRITIN
Anoxia Freezing cGMP cAMP Ca2 Ion
Ferritin Transcription
Anoxic control
Control
normoxia
A
B
a
anoxia
Anoxic
freezing
a
In other systems, ferritin heavy chain
transcription is induced by - Hypoxia
- cAMP - PMA
a
db-cGMP
a
db-cAMP
PMA
a
Ca2Ion
b
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ANTIOXIDANT ENZYMES
Willmore WG Storey KB - FEBS J 272, 3602-14
(2005) - Am J Physiol 273, R219-25 (1997).
- Mol Cell Biochem 170, 177-185 (1997)
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PRINCIPLES OF HYPOXIA SURVIVAL
1. Metabolic rate depression 2. Alternative end
products 3. Reversible phosphorylation of
enzymes 4. Overall suppression of
transcription translation 5. Selected genes
up-regulated
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ANOXIA SURVIVAL
Lutz PL Milton SL. 2004. J Exp Biol 207
3141-3147
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GENES
Control by transcriptional regulation
Transcription
RNAs
Control by translational regulation
Translation
Control by proteases
No Modification
PROTEINS (ENZYMES)
INACTIVE ENZYME
Degradation
Covalent modification
Control by post- translational modification
FUNCTIONAL ENZYMES
Inhibition and Activation
Control at level of enzyme function
ACTIVE ENZYMES
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ANOXIA

• J. STOREY
• S. BROOKS
• Q. CAI
• W. WILLMORE
• H. MEHRANI
• D. DOUGLAS
• J. DUNCAN
• S. GREENWAY

• M. HERMES-LIMA
• T. ENGLISH
• K. LARADE
• E. RUSSELL
• T. PANNUNZIO
• R. WHITWAM
• S. KORYCAN
• B. MICHAELIDIS

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PRINCIPLES OF HYPOXIA SURVIVAL
1. Metabolic rate depression 2. Overall
suppression of transcription
translation 3. Alternative end products4.
Reversible phosphorylation of enzymes5.
Selected genes up-regulated
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OXYRADICAL DAMAGE TO PROTEINS
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Anatomy of Littorina littorea
Foot
Hepatopancreas
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Protein to GWW ratio
7
7
8
8
3
3
6
6
1
1
5
5
4
2
2
4
Normoxia
48 Anoxia
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Isolated Organ Culture
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PRINCIPLES OF METABOLIC ARREST

• 1. Metabolic rate reduction
• 2. Control by protein kinases(SAPKs, 2nd
  messenger PKs)
• 3. Selective gene activation

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Protein Synthesis
Incorporation of 3H (cpm x 103/mg/h)
a
a
a
a
Recovery
Anoxia
Con 24h 1R
eIF-2a
eIF-2a-P
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ANOXIA INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Gene inactivation ( mRNA )
• Few Genes activated

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ANOXIA INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Gene inactivation ( mRNA )
• Few Genes activated

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