SALMAN UL ISLAM
19-JUNE-2014
Kyungpook National University
Contents
• Background information
• Aim of research
• Strategy
• Materials and methods
• Results
• Summary
BACKGROUND
INFORMATION
Pannexins are four-pass
transmembrane channels.
 PANX1 is the most widely
expressed among the
pannexin family members
(P...
PANX1 is a target of effector caspase cleavage during
apoptosis
Trovafloxacin
o It is a “Fourth Generation” flouroquinolone antibiotic. It is
bactericidal in nature.
Trovafloxacin
Generation Drug Names Spectrum
1st
Nalidixic acid
Cinoxacin
Gram- but not Pseudomonas
species
2nd
Norfloxacin
Ciprofloxaci...
Mode of action of Trovafloxacin
o Inhibition of bacterial DNA Gyrase (Topoisomerase II)
 Formation of quinolone-DNA-Gyras...
AIM OF RESEARCH
Aim Of Research
o To identify small molecules that can modulate PANX1
function.
STRATEGY
Strategy
o To confirm that Trovafloxacin inhibits pannexin 1 activity during apoptosis:
 Induction of apoptosis by activa...
MATERIALS
&
METHODS
Materials & methods
o LOPAC 1280: Product by Sigma Aldrich---having 1280
pharmacologically active molecules---collection o...
Materials & methods
o Anti-Fas antibody: The antibody demonstrates cytolytic
activity on human cells that express Fas. Cel...
Materials & methods
o TO-PRO-3: An excellent far red-fluorescent nuclear and chromosome
counterstain---impermeant to live ...
Materials & methods
o TEV(Tobacco Etch Virus) Protease: Cysteine protease
from Tobacco Etch Virus--- strict 7 amino acid c...
Materials & methods
o Patch Clamp Technique: Electrophysiological technique---
allows the study of single or multiple ion ...
Materials & methods
Materials & methods
o Time lapse microscopy: Live cell microscopy---to see the cells
in action.
Global knock out mice.
Cre-lox recombination
o Cre recombinase: Recombinase enzyme---derived from
Bacteriophage P1---catalyse the site specific r...
34bp lox p site
Conditional knockout mice
RESULTS
Fig. 1 Trovafloxacin inhibits pannexin 1 activity during apoptosis.
Fig. 2 Trovafloxacin-mediated inhibition of PANX1 promotes
formation of smaller apoptotic bodies.
Fig. 3 Pannexin 1 regulates disassembly of apoptotic thymocytes.
Fig. 4 Formation of string-like apoptopodia after membrane blebbing
correlates with formation of apoptotic bodies.
http://...
SUMMARY
Summary
• During apoptosis, PANX1 channels are cleaved by caspases
which then relaese “find me’’ signal to recruit phagocy...
Best wishes for all of you!
Unexpected link between an antibiotic, pannexin channels and apoptosis.
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Unexpected link between an antibiotic, pannexin channels and apoptosis.

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Unexpected link between an antibiotic, pannexin channels and apoptosis.

  1. 1. SALMAN UL ISLAM 19-JUNE-2014 Kyungpook National University
  2. 2. Contents • Background information • Aim of research • Strategy • Materials and methods • Results • Summary
  3. 3. BACKGROUND INFORMATION
  4. 4. Pannexins are four-pass transmembrane channels.  PANX1 is the most widely expressed among the pannexin family members (PANX1, PANX2, PANX3).  PANX1 can conduct molecules up to 1kDa in size across the plasma membrane (ATP and UTP are 507 and 484 daltons, respectively). *UTP= Source of energy like ATP. PANNEXINS
  5. 5. PANX1 is a target of effector caspase cleavage during apoptosis
  6. 6. Trovafloxacin o It is a “Fourth Generation” flouroquinolone antibiotic. It is bactericidal in nature. Trovafloxacin
  7. 7. Generation Drug Names Spectrum 1st Nalidixic acid Cinoxacin Gram- but not Pseudomonas species 2nd Norfloxacin Ciprofloxacin Enoxacin Ofloxacin Gram- (including Pseudomonas species), some Gram+ (S. aureus) and some atypicals 3rd Levofloxacin Sparfloxacin Moxifloxacin Gemifloxacin Same as 2nd generation with extended Gram+ and atypical coverage 4th *Trovafloxacin Same as 3rd generation with broad anaerobic coverage Classification of Flouroquinolones
  8. 8. Mode of action of Trovafloxacin o Inhibition of bacterial DNA Gyrase (Topoisomerase II)  Formation of quinolone-DNA-Gyrase complex  Induced cleavage of DNA---rapid death of bacterial cells.It unwinds the DNA supercoils by cutting and resealing the single DNA strand
  9. 9. AIM OF RESEARCH
  10. 10. Aim Of Research o To identify small molecules that can modulate PANX1 function.
  11. 11. STRATEGY
  12. 12. Strategy o To confirm that Trovafloxacin inhibits pannexin 1 activity during apoptosis:  Induction of apoptosis by activating Fas Pathway using Anti-Fas antibody.  Confirmation of opening of Pannexin 1 channels----- TO-PRO-3 take up by apoptotic cells.  Measurement of apoptosis-induced plasma membrane PANX1 currents at the single cell level. o To confirm that PANX1 inhibition by Trovafloxacin promotes formation of smaller apoptotic bodies:  Time lapse microscopic imaging of apoptotic cells.  Flow cytometric analysis using fluorescent dyes. o To confirm that PANX1 regulates disassembly of apoptotic thymocytes:  Induction of apoptosis by Dexamethasone in thymocytes; ex vivo and in vivo.  In vivo Testing of Trovafloxacin in thymic apoptosis.  Generation of PANX1 global and conditional knockout mice.  Time lapse microscopy & flow cytometry of panx1 knockout thymocytes. o To confirm that apoptodia formation is correlated with apoptotic bodies formation:  Induction of apoptosis.  Pharmacological inhibition and genetic modification of PANX1.  Time lapse microscopic and flowcytometric analysis of cells undergoing apoptosis.
  13. 13. MATERIALS & METHODS
  14. 14. Materials & methods o LOPAC 1280: Product by Sigma Aldrich---having 1280 pharmacologically active molecules---collection of inhibitors, receptor ligands, pharma-developed tools, and approved drugs---impacts most signaling pathways and covers all major drug target classes.
  15. 15. Materials & methods o Anti-Fas antibody: The antibody demonstrates cytolytic activity on human cells that express Fas. Cells undergo apoptosis in response to this antibody.
  16. 16. Materials & methods o TO-PRO-3: An excellent far red-fluorescent nuclear and chromosome counterstain---impermeant to live cells but penetrates compromised membranes characteristic of dead cells---useful indicator of dead cells within a population o ANNEXIN-V : A phosphatidylserine (PS) binding protein---used to detect PS on the surface of cells undergoing apoptotis.
  17. 17. Materials & methods o TEV(Tobacco Etch Virus) Protease: Cysteine protease from Tobacco Etch Virus--- strict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln↓Gly. o Dexamethasone: Glucocorticoid---causes thymic and splenic involution in rodents through apoptosis. (Immunology and Cell Biology (2000) 78, 238–246; doi:10.1046/j.1440-1711.2000.00905.)
  18. 18. Materials & methods o Patch Clamp Technique: Electrophysiological technique--- allows the study of single or multiple ion channels in cells.
  19. 19. Materials & methods
  20. 20. Materials & methods o Time lapse microscopy: Live cell microscopy---to see the cells in action.
  21. 21. Global knock out mice.
  22. 22. Cre-lox recombination o Cre recombinase: Recombinase enzyme---derived from Bacteriophage P1---catalyse the site specific recombination event between two lox P sites. o Lox P site: 34bp sequence---also derived from Bacteriophage P1---specific site for cleavage by Cre recombinase enzyme. o Floxing: It refers to the sandwiching of a Gene between two lox P sites---it is then said to be floxed gene.
  23. 23. 34bp lox p site
  24. 24. Conditional knockout mice
  25. 25. RESULTS
  26. 26. Fig. 1 Trovafloxacin inhibits pannexin 1 activity during apoptosis.
  27. 27. Fig. 2 Trovafloxacin-mediated inhibition of PANX1 promotes formation of smaller apoptotic bodies.
  28. 28. Fig. 3 Pannexin 1 regulates disassembly of apoptotic thymocytes.
  29. 29. Fig. 4 Formation of string-like apoptopodia after membrane blebbing correlates with formation of apoptotic bodies. http://www.nature.com/nature/journal/v507/n7492/full/nature13147.html#videos
  30. 30. SUMMARY
  31. 31. Summary • During apoptosis, PANX1 channels are cleaved by caspases which then relaese “find me’’ signal to recruit phagocytes. • Trovafloxacin inhibits PANX1 channels leading to formation of smaller apoptotic disassembled, less granular cells. • Genetic modification of PANX1 is phenocopoying the Trovafloxacin effects. • Unique structure of Trovafloxacin may be responsible for its unexpected effects, and further study on this may unlock the clue which will lead to production of safer and more potent antibiotics.
  32. 32. Best wishes for all of you!

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