Antti Pajunen

Ornithine decarboxylase and adenosylmethionine decarboxylase in mouse brain--effect of electrical stimulation.

THE POLYAMINES. spermidine and spermine, and the diamine. putrescine, have received considerable attention in recent years as they have been implicated in various growth processes and in cellular differentiation (RAINA & JANNE, 1975). They occur in high concentrations in neural tissue (SHASKAN eta/., 1973; SEILER & SCHMIDT-GLENEWINKEL, 1975; SHAW & PATEMAN. 1973; HARIK & SNYDER, 1974) and show a distinct developmental pattern and age dependency (JANNE el al., 1964; KREMZNER et al., 1970). Ornithine decarboxylase (EC 4.1.1.17) catalyzes the conversion of L-ornithine to the diamine putrescine, the first and perhaps rate-limiting step in polyamine biosynthesis. S-Adenosyl-~-methionine decarboxylase (EC 4. I . 1.50) forms S-methyladenosylhomocysteamine from adenosylmethionine and spermidine synthase transfers the propylamino group from decarboxylated adenosylmethionine to putrescine. The spermine synthase reaction is analogous, transferring the propylamino group from decarboxylated adenosylmethionine to spcrmidine, forming spermine. The synthesis of polyamines has been extensively studied during past 10 years, but relatively little is known about their catabolism. Especially relevant to the function of the nervous system there is an important route to catabolize putrescine to GABA and further either to COz via the tricarboxylic cycle (SEILER et a)., 1971) or homocarnosine (KALYAKKER & MEISTER. 1959; KONISHI et a/., 1977). In this paper we have investigated the eficct of electrical stimulation of mouse brain on the activity of ODC and SAM-DC.

Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

Mouse brain ornithine decarboxylase activity is about 70‐fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immuno‐reactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme‐mediated mechanism in adult brain.

Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study

SummaryAnesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratiom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P≤0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress.

THE EFFECT OF dl‐ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN

Abstract— dl‐Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l‐ornithine decarboxylase activity and a simultaneous decrease in S‐adenosyl‐l‐methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl‐allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl‐allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.

Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-beta-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of beta-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

ISCHEMIA - INDUCED DISTURBANCES OF POLYAMINE SYNTHESIS

Publisher Summary This chapter discusses ischemia-induced disturbances in polyamine synthesis. It discusses the periods of time during which changes take place and the relationship of changes in polyamine synthesis to the duration of ischemia. Pharmacological interventions reducing ischemia-induced disturbances in polyamine synthesis are discussed. Emphasis is put on the possible role of ischemia-induced alterations in polyamine metabolism in the process of recovery from the metabolic stress produced either by cerebral ischemia or in the manifestation of neuronal necrosis. Transient cerebral ischemia causes a disturbance of polyamine synthesis that is characterized by a sharp increase in ornithine decarboxylase and decrease in S-adenosylmethionine decarboxylase activity. The result is an overshoot in putrescine formation and a reduction in spermine levels. In addition, evidence is presented that polyamines are released from the intracellular compartment during ischemia and following prolonged recirculation in severely damaged areas. Because of the main features of ischemia-induced disturbances in polyamine synthesis and the reactions known to be influenced by these altered polyamine profiles it is suggested that these changes play a role in the manifestation of ischemia-induced neuronal necrosis.

Changes in gene expression in response to polyamine depletion indicates selective stabilization of mRNAs.

We used differential display analysis to identify mRNAs responsive to changes in polyamine synthesis. As an overproducing model we used the kidneys of transgenic hybrid mice overexpressing ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine biosynthesis. To identify mRNAs that respond to polyamine starvation, we treated Rat-2 cells with alpha-difluoromethylornithine, a specific inhibitor of polyamine biosynthesis. We isolated 41 partial cDNA clones, representing 37 differentially expressed mRNAs. Of these, 15 have similarity with known genes, five appear to be similar to reported expressed sequence tags and seventeen clones were novel sequences. Of the 35 mRNAs expressed differentially after alpha-difluoromethylornithine treatment, 26 were up-regulated. The expression of only three mRNAs was altered in the transgenic animals and all three were down-regulated. Determination of mRNA half-life of three of the mRNAs up-regulated in response to polyamine depletion revealed that the accumulation results from stabilization of the messages. Because most of the transcripts identified from Rat-2 cells suffering polyamine starvation were accumulated, we conclude that polyamine depletion, while blocking cell growth, is stabilizing mRNAs. This may be due to the lack of spermidine for post-translational modification of the eukaryotic initiation factor 5A, which plays a major role in mRNA turnover. The coupling of mRNA stabilization with cell-growth arrest in response to polyamine starvation provides cells with an economical way to resume growth after recovery from polyamine deficiency.

Immunohistochemical localization of l-Ornithine decarboxylase in developing rat brain

l‐Ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis and a marker enzyme of tissue proliferation and maturation, was localized immunocytochemically in the developing rat central nervous system. It can be noted that the distribution of the enzyme protein underlies temporal alterations. Conclusions are drawn from the location of the enzyme and possible functional roles played by ornithine decarboxylase in discrete brain areas.

Expression of cytosolic acetyl-CoA synthetase gene is developmentally regulated.

Acetyl-CoA synthetase (AceCS) provides acetyl-CoA for different physiological processes, such as fatty acid and cholesterol synthesis, as well as the citric acid cycle. We show here that the cytosolic isoform of this enzyme, AceCS1, is expressed during mouse development. In the embryonic stage E9.5 AceCS1 transcripts localize in the cephalic region. At E10.5 the cephalic expression intensifies and transcripts appear also in the spinal cord and in the dorsal root ganglions. During organogenesis AceCS1 is expressed in the liver from E11.5. The AceCS1 gene is expressed also in the testes from E12.5 onwards and expression localizes in the interstitial Leydig cells. In the ovaries, expression is transient and AceCS1 transcripts are detected from E13.5 to E15.5 in the ovarian interstitial component. In the kidneys AceCS1 transcripts appear in a subset of the renal tubules at E16.5 and remains in these structures in newborns. Hence, expression of AceCS1 is developmentally regulated suggesting a role for AceCS1 during embryogenesis.

Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts from rat prostate

Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.