THE CHARACTERIZATION OF ANTIZYME IN DEVELOPING Xenopus laevis EMBRYOS

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THE CHARACTERIZATION OF ANTIZYME IN DEVELOPING
Xenopus laevis EMBRYOS

• Alexandra Silveira and Charles Toth Department of
  Biology
• Providence College, Providence, RI

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Protein Degradation

• Cell cycle and proliferation
• Differentiation and development
• Stress response
• Genesis of neural networks
• Modulation of cell surface receptors
• Regulation of immune response
• Apoptosis
• Transcriptional regulation
• Pathogenesis
• Cancer, cystic fibrosis, neurodegenerative
  diseases

Charles Toth, Providence College
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Protein degradation by the 26S proteasome
http//www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/
part1/protease.htm
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Protein degradation by the 26S proteasome
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ODC degradation a unique protein degradation
pathway
John Mitchell, University of Northern Iowa
John Mitchell, University of Northern Iowa
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ODC and Polyamine Biosynthesis
http//www.bios.niu.edu/mitchellab/general.html
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Polyamines

• Organic compounds found bound to RNA and DNA in
  cells
• High levels are toxic
• Necessary for cell growth and survival
• gate currents of ion channels
• have effects on chromatin condensation and
  transcriptional regulation
• neutralize negative charges of RNA and DNA
• regulate the levels of AZ
• provide a necessary postranslational modification
  to eIF-5a protein

http//www.biol.lu.se/zoofysiol/Cellprolif/Researc
h/research_area_1.html
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AZ Functions

• ODC degradation
• Anti-proliferative effect on cells
• Blocks uptake of additional polyamines when
  polyamine levels are too high
• High levels of polyamines in turn regulate AZ
  through a ribosomal frameshift

http//www.bios.niu.edu/mitchellab/general.html
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Ribosomal Frame-Shifting

• High levels of polyamines cause a ribosomal frame
  shift that produces an active form of AZ.
• Without the ribosomal shift a truncated and
  inactive AZ is formed.

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Why study antizyme?

• Studying the unique ODC degradation pathway can
  lead to a better understanding of
• ubiquitin mediated protein degradation
• the many roles of polyamines in the cell
• AZ is a member of an ancient gene family
• AZ1 and AZ2 widely expressed and involved in ODC
  degradation
• AZ3 is testis specific

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Why study antizyme?

• AZ has been cloned from various organisms
• vertebrate-man, chicken, rat, mouse, zebrafish,
  Xenopus, electric ray
• invertebrate-Drosophila, S. pombe, C. elegans,
  silk worms, gray mold, etc.
• AZ knockout in mice (Matsufuji Noda)
• viable and morphologically normal
• high rate of prenatal fatality from high levels
  of ODC
• phenotype complicated by additional family
  members

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Xenopus As A Developmental Model

• Ideal model because
• easily cared for in lab
• in vitro fertilization
• the developing embryos are easily cared for
• the embryos develop quickly

http//www.xenopus.com/products.htm
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Xenopus life cycle
Wolpert et al., Development 2000
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Methods

• In vitro fertilization
• Northern Blot
• Immunohistochemical Staining

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In Vitro Fertilization

• Female frogs are injected with HCG to induce
  ovulation
• Male frogs are sacrificed in order to obtain
  internal testicles
• Eggs are fertilized in vitro

http//www.fundamentalbiology.arc.nasa.gov/Image1.
jpg
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Northern blot analysis
www.columbia.edu/.../c2005/handouts/
northernforweb.gif
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Northern blot results

• AZ is expressed as a maternal and zygotic
  transcript
• Size of message increases post-MBT
• MBT is the point where zygotic genes are
  expressed instead of maternal genes and the cell
  cycle becomes asynchronous indicating the
  beginning of differentiation

Charles Toth, Providence College
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Conclusions

• Different sizes of antizyme mRNA are present
  during different times in embryonic development.
• A larger mRNA message is present post-MBT.

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Possible Implications

• The larger messages can indicate a
• a closely related familial gene
• different post-transcriptional modifications that
  ensure zygotic gene expression
• Results are inconclusive because of the ribosomal
  frame-shift needed to produce an active form of
  AZ.

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Immunohistochemical Staining

• Antibodies against antizyme are used to
  detect and stain antizyme in the developing
  embryo.

http//www-celanphy.sci.kun.nl/Bruce20web/Bruce2
0Gifs/immunocyto.gif
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Immunohistochemical Staining Results

• Stage 10 embryos
• There is a darkly staining ring around the
  blastopore from which the mesoderm will develop.
• The animal cap is almost uniformly stained.

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Immunohistochemical Staining Results

• Stage 18/19
• Top image
• staining along the neural groove and at both the
  anterior and posterior ends of the embryo, some
  staining at the ventral side
• Bottom image
• dorsal side shows dark staining at the posterior
  and anterior ends and a ring pattern of staining
  in the area that will eventually give rise to the
  mesoderm

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Immunohistochemical Staining Results

• Stage 33/34
• Staining is primarily focused around the somites
  and the anterior end of the tadpole
• These locations are where the muscles and nerves
  are developing

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Conclusions

• There is clearly a differential expression of
  antizyme during the various stages of embryonic
  development.
• staining of ring around the blastopore and animal
  cap
• staining of the neural groove, anterior and
  posterior ends, ventral, and dorsal lateral
  staining
• staining of the somites and anterior portion of
  the tadpole

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Possible Implications

• The staining around the blastopore ring, the
  dorsal-lateral staining of the neurula, and the
  somite staining of the tadpole implicate a role
  of antizyme in muscle development.
• The staining of the animal cap and the anterior
  end of the developing embryo implicate a role of
  antizyme in the development of the central
  nervous system.

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Future Endeavors

• In situ
• Western
• ablation studies
• forced expression

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Acknowledgements

• Dr. Charles Toth
• Dr. Senya Matsufuji
• Jikei University, Tokyo
• Tiffany LaFortune
• My family
• My roommates