Barbara Grzelakowska-Sztabert

Agmatine modulates the in vivo biosynthesis and interconversion of polyamines and cell proliferation.

Agmatine has recently gained wide interest as a bioactive arginine metabolite with a multitude of physiological functions. This study evaluates the in vivo role of agmatine in the modulation of metabolism and intracellular level of polyamines. Here, we report that agmatine, administered to mice, differentially affects the renal and liver activity of the two key enzymes regulating polyamine biosynthesis and interconversion/degradation. Thus, agmatine exerts a negative regulation of ODC activity and protein content, and positive regulation of SSAT activity, having no effect on ODC and SSAT transcript level. Agmatine modulation of ODC and SSAT activities is noticeably augmented by the inhibitor of its catabolism, aminoguanidine. Antizyme and eIF4E protein content appears to be affected by agmatine only insignificantly and apparently do not contribute to agmatine-induced down-regulation of ODC content. The homeostasis of spermidine and spermine is preserved after agmatine injection, while the putrescine level decreases. Furthermore, when tested in a mouse kidney injury model, agmatine, partially but significantly, reduces [3H] thymidine incorporation into DNA. This is consistent with suppressed renal tubule epithelial cell proliferation. The findings provide in vivo evidence of a substantial role of agmatine as a modulator of polyamine biosynthesis and degradation and suggest its suppressive effect on cell proliferation.

Inhibitors of polyamine biosynthesis affect the expression of genes encoding cytoskeletal proteins.

The polyamines are ubiquitous components of mammalian cells. Those compounds have been postulated to play an important role in different cellular functions including the reorganization of cytoskeleton associated with the cell cycle. In the studies reported here, it was found that inhibitors of polyamine biosynthesis, methylglyoxal‐bis[quanylhydrazone] (MGBG) and difluoromethylornithine (DFMO), prevent mitogen‐induced accumulation of mRNAs encoding major cytoskeletal components, β‐actin and α‐tubulin, in mouse splenocytes. These findings suggest mechanisms through which polyamines may exert their effects on the cytoskeleton integrity.

Tissue-specific effects of testosterone on S-adenosylmethionine formation and utilization in the mouse

Exogenous administration of testosterone produced several metabolic tissue-specific changes in female mouse kidneys, but not in the liver. The hormone induced ornithine decarboxylase (ODC) activity, and also profoundly influenced metabolism of S-adenosylmethionine (AdoMet). Therefore, the activity of the AdoMet-synthesizing enzyme (AdoMet synthetase) and of cystathionine synthase, which commits homocysteine irreversibly to the transsulfuration pathway, were significantly increased. In contrast to the level of AdoMet in the liver the renal level of this metabolite was augmented, whereas the level of S-adenosylhomocysteine (AdoHcy) did not change. This resulted in an increase of the AdoMet/AdoHcy ratio. In testosterone-treated mice, pulse-labelled with [methyl-14C]methionine, the radioactivity recovered in the kidneys doubled, but in the liver remained the same. The rise in radioactivity recovered occurred mainly in TCA-soluble compounds and lipids, and to a smaller extent, in proteins and nucleic acids.

Arginine and ornithine metabolizing enzymes in testosterone-induced hypertrophic mouse kidney

Administration of testosterone to female mice causes hypertrophy of their kidneys with spectacular induction of ornithine decarboxylase and significant increase in the level of putrescine. We tried to find out whether testosterone treatment affects also the renal activities of enzymes participating in the formation and utilization of ornithine, specifically arginase and ornithine aminotransferase, and whether they are dependent on putrescine level. Swiss, CFW, DBA2 or F1 (CFW x DBA2) female and male mice were injected with testosterone (125 mg/kg) or CB 3717 (100 mg/kg). DFMO was applied in the drinking water. The activities of the enzymes were determined 24 hr or 5 days after administration of CB 3717 or testosterone, respectively. Renal activities of ornithine decarboxylase (ODC), arginase and ornithine aminotransferase (OAT) were found to be sex-differentiated. The highest activity of ODC was characteristic for the kidneys of males, whereas those of arginase and OAT for the kidneys of females. In the kidneys of testosterone-treated female mice a decrease (50%) of OAT, and a significant increase of arginase activities (up to 200%), were observed. In the males these changes were less pronounced. DFMO, which completely inhibited the activity of renal ODC, did not influence significantly the testosterone-induced arginase and the testosterone-decreased OAT. Arginase and OAT, in contrast to ODC, were not changed in CB 3717-induced hyperplastic kidney. The study showed testosterone-induced differential changes in the activity of two enzymes involved in ornithine biosynthesis and catabolism which accompanied ODC induction in female mouse kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of G0-S transition in mouse T lymphocytes by polyamines.

While the role of polyamines in DNA synthesis during the S phase of the cell cycle has been repeatedly postulated, recent studies point also to polyamine involvement in the early phase of the G0-S transition. In order to determine polyamine-dependent steps in the cell cycle we have studied the effects of inhibitors of polyamine biosynthesis and exogenous polyamines on the proliferation of T lymphocytes as well as on the expression of some growth-regulated genes. The ability of Con A-stimulated mouse T lymphocytes to enter DNA synthesis was markedly inhibited by methylglyoxal bis(guanylhydrazone) in a dose-dependent manner. This inhibitory effect was stronger in the presence of fetal calf serum containing a high level of activities of polyamine oxidases than in the presence of horse serum. Putrescine and spermine added to T splenocyte culture instead of mitogen-Con A stimulated [3H]thymidine incorporation with kinetics similar to that observed with Con A. The growth-stimulating effects of polyamines were concentration-dependent. Polyamines at optimal growth-stimulating concentrations (10 microM spermine and 80 microM putrescine) induced the expression of genes encoding the cytoskeletal proteins beta-actin, vimentin, and alpha-tubulin to an extent and with kinetics similar to those of Con A. The results presented herein suggest that polyamines are capable of stimulating the transition of G0 cells to the S phase and that this effect may be mediated by their influence on the gene expression.

Polyamines in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney. Differential effect of α-difluoromethylornithine

In the testosterone-induced hypertrophic and antifolate (N10-propargyl,5,6-dideazafolic acid, CB 3717)-induced hyperplastic mouse kidney models, a marked increase of two diamine levels--putrescine and cadaverine--occurred which paralleled induced ornithine decarboxylase (ODC) activity. Under these conditions the augmentation of spermidine levels was much smaller, while spermine levels were affected differentially--increased by testosterone and decreased by CB 3717; this resulted in an increase of spermidine/spermine ratio in hyperplastic, but not hypertrophic kidney. alpha-Difluoromethylornithine (DFMO) prevented testosterone- or CB 3717-induced increment of both diamine levels. Spermidine and spermine depletion in response to DFMO was significant in hyperplastic kidney only. DFMO also significantly affected the other biochemical markers of hyperplasia, namely lowered CB 3717-induced cell proliferation rate and increased S-adenosylmethionine decarboxylase (AdoMetDC) activity. In contrast, testosterone-induced hypertrophy was not influenced by DFMO, as judged by the lack of its effect on S-adenosylmethionine synthetase and cystathionine and synthase activity. These results indicate that the increase of putrescine levels does not mediate testosterone-induced renal hypertrophy and possibly also antifolate-induced hyperplasia. The involvement of spermidine in mediation of renal hyperplasia is highly possible, while that of spermine is excluded.

Induction of betaine: Homocysteine methyltransferase in some murine cells cultured in vitro

Betaine when present in the culture medium could induce the activity of betaine: homocysteine methyltransferase (EC 2.1.1.5) in mouse L-cells, and leukemic L1210 cells, as well as in mouse embryo fibroblasts grown in vitro. We found this process to be time- and concentration-dependent. A persisting contact of the cells with betaine was indispensible for expressing and maintaining the enzyme activity. The treatment of cells with cycloheximide or actinomycin D abolished the process of induction. Methionine as well as homocysteine, when present either in the culture medium or in the reaction mixture, strongly depressed the activity of this enzyme. The L-cells with the induced betaine:homocysteine methyltransferase survived but did not multiply in the methionine-deficient medium, therefore, they did not become prototrophs with respect to methionine.

The transfer of one-carbon units in insect metabolism. Pathways of folate coenzyme synthesis

Abstract Enzymes involved in the reduction of folate to tetrahydrofolate and in binding the one carbon-units to tetrahydrofolate have been investigated in preparations of insect tissues. In the two-step reduction of folate to tetrahydrofolate, dihydrofolate appears to be the preferred substrate for the oxidoreductases extracted from Galleria mellonella . The enzyme using formate as a C 1 source is much more active than the enzyme system using the hydroxymethyl group of serine. No enzyme system which would catalyse the binding of the formimino group either from formiminoglutamate or formiminoglycine was found.

NUCLEAR FACTOR OF ACTIVATED T CELLS (NFAT) IS A POSSIBLE TARGET FOR DEXAMETHASONE IN THYMOCYTE APOPTOSIS

Cell death plays a critical role in the generation of an effective immune system. During maturation T lymphocytes are generated, censored and eliminated in the thymus. These events are temporally associated with developmental changes in the levels of transcription factors including NFAT. The NFAT transcription factor (nuclear factor of activated T cells) is implicated in the regulation of T‐lymphocyte proliferation and transcriptional activation of genes encoding lymphokines. It has been demonstrated that discontinuities in the inducibility of NFAT and AP‐1 transcription factors occur during transition of immature thymocytes into cortical thymocytes which are eliminated by apoptosis.

Catecholamines are required for androgen-induced ODC expression but not for hypertrophy of mouse kidney.

Catecholamine depletion, evoked by reserpine, dramatically impaired (5-fold) the testosterone-induced increase of ornithine decarboxylase (ODC) activity in female mouse kidney. However, reserpine did not prevent kidney hypertrophy evoked by testosterone. This is evidenced by the activity of sensitive, biochemical markers of renal hypertrophy, namely arginase and ornithine aminotransferase (OAT), that responded with the increase and decrease of activities to testosterone treatment, respectively. Arginine and ornithine, substrates and/or products of marker enzymes, showed a striking homeostasis as their level was not affected by testosterone and reserpine, and only slightly by DFMO. Northern blot analysis revealed that the ODC mRNA level, that was increased 10-fold by testosterone, was decreased 2-fold in catecholamine-depleted hypertrophic kidney. Thus, ODC transcript level, lowered by reserpine, correlated partially with an attenuated response of ODC activity to testosterone. This was in contrast to DFMO, which inhibited ODC activity, but significantly increased its mRNA content. It is concluded that catecholamines could be involved together with testosterone in regulation of the ODC gene expression in mouse kidney.