Aki Järvinen

Animal disease models generated by genetic engineering of polyamine metabolism

The polyamines putrescine, spermidine and spermine are natural components of all living cells. Although their exact cellular functions are still largely unknown, a constant supply of these compounds is required for mammalian cell proliferation to occur. Studies with animals displaying genetically altered polyamine metabolism have shown that polymines are intimately involved in the development of diverse tumors, putrescine apparently has specific role in skin physiology and neuroprotection and the higher polyamines spermidine and spermine are required for the maintenance of pancreatic integrity and liver regeneration. In the absence of ongoing polyamine biosynthesis, murine embryogenesis does not proceed beyond the blastocyst stage. The last years have also witnessed the appearance of the first reports linking genetically altered polyamine metabolism to human diseases.

Neuroprotective role of ornithine decarboxylase activation in transient focal cerebral ischaemia: a study using ornithine decarboxylase-overexpressing transgenic rats

Nuclear magnetic resonance imaging (MRI) was used to study dynamics of maturation and the size of ischaemic stroke lesions in rats with greatly increased activity of ornithine decarboxylase (ODC). Syngenic rats, either with or without chronic pre‐ischaemic treatment with an ODC inhibitor, α‐difluoromethylornithine (DFMO), as well as ODC‐overexpressing transgenic rats were subjected either to transient middle cerebral artery (MCA) occlusion or permanent occlusion of the cortical branch of MCA. The two models were chosen to assess the role of ODC activity in damage caused by ischaemia and reperfusion, respectively. Diffusion of water was quantified by means of the trace of the diffusion tensor (Dav = Trace=D) to assess the extent of energy failure and cytotoxic oedema, whereas the spin–spin relaxation time (T2) was used as a quantitative indicator of irreversible damage by MRI. Exposure to transient MCA occlusion resulted in significantly smaller stroke lesions in the ODC‐overexpressing transgenic (246 ± 14 mm3) than in syngenic (320 ± 9 mm3) or DFMO‐treated (442 ± 63 mm3) rats as determined 48 h after the occlusion. The differences in sizes were due to smaller lesions in the cortical tissue (transgenic vs. syngenic) or both in cortical and striatal regions (transgenic vs. DFMO‐treated animals). The degree of irreversible oedema was greater in DFMO‐treated rats than in syngenic or transgenic animals indicating accelerated development of a permanent damage in the absence of ODC induction. Cortical infarct following permanent MCA occlusion developed faster in the DFMO‐treated than in syngenic or transgenic rats as the lesion sizes at 10 h were 26.2 ± 4.3 mm3, 14.2 ± 2.3 mm3 and 12.3 ± 1.9 mm3, respectively. However, the stroke volumes by 48 h were not statistically different in the three animal groups. The present data demonstrate that ODC activation is an endogenous neuroprotective measure in transient cerebral ischaemia.

Transgenic Rats as Models for Studying the Role of Ornithine Decarboxylase Expression in Permanent Middle Cerebral Artery Occlusion

BACKGROUND AND PURPOSE Cerebral ischemia causes activation of ornithine decarboxylase (ODC) gene and subsequent accumulation of putrescine, which might either directly or indirectly affect the outcome of cerebral infarct. We developed a transgenic rat overexpressing human ODC, which was used to explore the effect of abnormally high putrescine concentration in the brain on the infarct volume after permanent middle cerebral artery (MCA) occlusion. METHODS The transgenic rats were produced by the pronuclear injection technique with the use of cloned human ODC gene. The right MCA was permanently occluded through craniotomy. ODC activity and polyamines were assayed in the infarcted and contralateral hemispheres. MRI was used to quantify T2 relaxation time, apparent diffusion constant (ADC), and infarct volume, which was also determined by 2,3,5-triphenyltetrazolium chloride. RESULTS Permanent MCA occlusion resulted in extensive activation of ODC, which was approximately sevenfold greater than in syngenic animals at 20 hours after occlusion. Consequently, putrescine increased from approximately 10 and 230 pmol/mg to 160 and 410 pmol/mg in the infarcted hemisphere of syngenic and transgenic animals, respectively, but all the other polyamines were unchanged. This high putrescine in the transgenic rats did not influence infarct size evolution, as determined by MRI, T2, ADC, or the infarct volume by 2,3,5-triphenyltetrazolium chloride at 48 hours. CONCLUSIONS Data from the ODC transgenic rat model show that the development of brain infarct after permanent MCA occlusion was not influenced by extensive levels of putrescine, indicating that this endogenous amine is not involved in maturation and spread of stroke lesion in vivo. Thus, it seems that ODC activation reflects an endogenous adaptation of neural cells to a noxious stimulus that does not directly influence lesion development.

Guide Molecule-driven Stereospecific Degradation of α-Methylpolyamines by Polyamine Oxidase

FAD-dependent polyamine oxidase (PAO; EC 1.5.3.11) is one of the key enzymes in the catabolism of polyamines spermidine and spermine. The natural substrates for the enzyme are N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine. Here we report that PAO, which normally metabolizes achiral substrates, oxidized (R)-isomer of 1-amino-8-acetamido-5-azanonane and N1-acetylspermidine as efficiently while (S)-1-amino-8-acetamido-5-azanonane was a much less preferred substrate. It has been shown that in the presence of certain aldehydes, the substrate specificity of PAO and the kinetics of the reaction are changed to favor spermine and spermidine as substrates. Therefore, we examined the effect of several aldehydes on the ability of PAO to oxidize different enantiomers of α-methylated polyamines. PAO supplemented with benzaldehyde predominantly catalyzed the cleavage of (R)-isomer of α-methylspermidine, whereas in the presence of pyridoxal the (S)-α-methylspermidine was preferred. PAO displayed the same stereospecificity with both singly and doubly α-methylated spermine derivatives when supplemented with the same aldehydes. Structurally related ketones proved to be ineffective. This is the first time that the stereospecificity of FAD-dependent oxidase has been successfully regulated by changing the supplementary aldehyde. These findings might facilitate the chemical regulation of stereospecificity of the enzymes.

α-Methylated Polyamines as Potential Drugs and Experimental Tools in Enzymology

We describe synthesis of alpha-methylated analogues of the natural polyamines and their use as tools in unraveling polyamine functions. Experiments with alpha-methylated spermidine and spermine revealed that the polyamines are exchangeable in supporting cellular growth. Degradation of the analogues by polyamine oxidase disclosed hidden, aldehyde-guided stereospecificity of the enzyme.

New syntheses of α-methyl- and α,α′-dimethylspermine

Abstractα-Methylspermine and α,α′-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.

A New Synthesis of α-Methylspermidine

A five-step synthesis of α-methylspermidine (1,8-diamino-5-azanonane), the first polyamine analogue preventing pathological consequences of spermidine depletion in transgenic rats overproducing spermine/spermidine N1-acetyltransferase, from ethyl 3-aminobutyrate was achieved in a high overall yield.

Genetic Engineering of Polyamine Catabolism in Transgenic Mice and Rats

The biosynthetic pathway of the polyamines is practically irreversible because it involves two decarboxylation reactions. The first evidence, however, indicating that spermidine can be converted to putrescine and spermine to spermidine emerged in the late 1960s when Siimes (1) found that radioactive spermidine and spermine yielded labeled putrescine and spermidine in rat liver in vivo. An additional 10 yr were required before the first enzyme, polyamine oxidase (PAO), of a separate polyamine backconversion pathway was purified and characterized (2). Polyamine oxidase strongly favors acetylated spermidine and spermine over the natural polyamines as its substrates (2). That the oxidation of unmodified spermidine and spermine is greatly enhanced by benzaldehyde (or other aldehydes) is in all likelihood attributable to Schiff base formation between the primary amino groups of the polyamine and the aldehydes, thus mimicking the charge distribution of acetylated polyamines (2). It soon became evident that spermidine and spermine are acetylated by a cytosolic enzyme, spermidine/spermine N 1 -acetyltransferase (SSAT), that is highly inducible and has a very short half-life (3). Recently, another oxidase involved in polyamine catabolism was found and named spermine oxidase (4,5). The latter enzyme is practically specific for spermine and does not catalyze the oxidation of spermidine or acetylated polyamines (4,5). In the SSAT/PAO-dependent backconversion pathway, SSAT clearly is the rate-controlling enzyme because PAO is a constitutively expressed enzyme occurring in great excess in comparison with inducible SSAT (6). Moreover, PAO oxidizes only acetylated polyamines.