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Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy.

ABSTRACT: Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac alpha-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10-39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30-32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.

SUBMITTER: Mackintosh CA

PROVIDER: S-EPMC1221293 | biostudies-other | 2000 Sep

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Ornithine decarboxylase antizymes are proteins which negatively regulate cellular polyamine levels via their affects on polyamine synthesis and cellular uptake. In virtually all organisms from yeast to mammals, antizymes are encoded by two partially overlapping open reading frames (ORFs). A +1 frameshift between frames is required for the synthesis of antizyme. Ribosomes change translation phase at the end of the first ORF in response to stimulatory signals embedded in mRNA. Since standard sequence analysis pipelines are currently unable to recognise sites of programmed ribosomal frameshifting, proper detection of full length antizyme coding sequences (CDS) requires conscientious manual evaluation by a human expert. The rapid growth of sequence information demands less laborious and more cost efficient solutions for this problem. This manuscript describes a rapid and accurate computer tool for antizyme CDS detection that requires minimal human involvement. RESULTS: We have developed a computer tool, OAF (ODC antizyme finder) for identifying antizyme encoding sequences in spliced or intronless nucleic acid sequenes. OAF utilizes a combination of profile hidden Markov models (HMM) built separately for the products of each open reading frame constituting the entire antizyme coding sequence. Profile HMMs are based on a set of 218 manually assembled antizyme sequences. To distinguish between antizyme paralogs and orthologs from major phyla, antizyme sequences were clustered into twelve groups and specific combinations of profile HMMs were designed for each group. OAF has been tested on the current version of dbEST, where it identified over six thousand Expressed Sequence Tags (EST) sequences encoding antizyme proteins (over two thousand antizyme CDS in these ESTs are non redundant). CONCLUSION: OAF performs well on raw EST sequences and mRNA sequences derived from genomic annotations. OAF will be used for the future updates of the RECODE database. OAF can also be useful for identifying novel antizyme sequences when run with relaxed parameters. It is anticipated that OAF will be used for EST and genome annotation purposes. OAF outputs sequence annotations in fasta, genbank flat file or XML format. The OAF web interface and the source code are freely available at http://recode.ucc.ie/oaf/ and at a mirror site http://recode.genetics.utah.edu/oaf/.

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Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy.

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