Effects of female sex hormones on polyamine-oxidizing enzyme activities and polyamine concentrations in immature rat uterus and liver

17-estradiol (E₂) and progesterone (P) treatment of immature female rats (10 g/100 g body weight) respectively resulted in 1.38-fold (p<0.02) and 1.42-fold (p<0.02) increase in the uterine polyamine oxidase activity, and 2.45-fold (p<0.001) and 1.43-fold (p<0.02) increase in the uterine diamine oxidase activity, as compared to the controls. E₂ caused a 5-fold (p<0.05) and a 1.36-fold (p<0.05) increase in putrescine and spermidine concentration respectively in rat uterus. Increases of 1.7-fold (p<0.02) and 1.6-fold (p<0.05) in putrescine and spermine concentration were determined in the P-treated uterus, as compared to the controls. The spermidine/spermine ratio, which is regarded as an index of growth rate, was higher in the E₂-treated uterus and lower in the P-treated uterus than in the control uterus. No statistically significant hormonal effects were estimated in the immature liver. The data reported suggest the possibility of an involvement of polyamine-oxidizing enzymes in the modulation of polyamine concentrations in rat uterus by the female sex hormones.     

Inhibition ofDugesia tigrina auricle regeneration by inhibitors of polyamine synthesis

Summary Results are presented that indicate polyamine synthesis inhibitors increase the flatwormDugesia tigrina's auricle regeneration time. This study serves as evidence that endogenous putrescine, spermidine, and spermine are necessary for the flatworm regeneration process.     

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC₅₀ values of 4.35 and 6.47 μM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 μM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N¹-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H₂O₂ and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis. Received 17 January 2002; received after revision 22 February 2002; accepted 22 February 2002     

Effect of inhibitors of polyamine biosynthesis on primary differentiation of mouse egg]

Alpha-Methylornithine, an inhibitor of the synthesis of putrescine does not affect the cleavage of Mouse eggs, cultured in vitro from the 2-cell stage, before blastocyst formation, whereas methylglyoxal-Bis (guanylhydrazone), an inhibitor of the syntheses of spermidine and spermine induces the embryos to become quiescent at about the 8-cell stage; resumption of their development, after transfer to fresh medium, is followed by a delay in cavitation. These results may be related to the biological clock theory for primary differentiation.     

Stimulation ofDugesia tigrina auricle regeneration by exogenous putrescine,spermine or spermidine

Summary Specimens of a flatworm,Dugesia tigrina were decapitated and then cultured in a solution of 1×10^–4 M putrescine, spermine or spermidine. Subsequent observation for the reappearance of auricles indicates that the amine treatment stimulates the flatworm regeneration process.     

Polyamine-dependent gene expression

The polyamines spermidine and spermine along with the diamine putrescine are involved in many cellular processes, including chromatin condensation, maintenance of DNA structure, RNA processing, translation and protein activation. The polyamines influence the formation of compacted chromatin and have a well-established role in DNA aggregation. Polyamines are used in the posttranslational modification of eukaryotic initiation factor 5A, which regulates the transport and processing of specific RNA. The polyamines also participate in a novel RNA-decoding mechanism, a translational frameshift, of at least two known genes, the TY1 transposon and mammalian antizyme. Polyamines are crucial for their own regulation and are involved in feedback mechanisms affecting both polyamine synthesis and catabolism. Recently, it has become apparent that the polyamines are able to influence the action of the protein kinase casein kinase 2. Here we address several roles of polyamines in gene expression.Received 27 November 2002; received after revision 9 January 2003; accepted 31 January 2003     

Regional pattern and heat-resistance of brain 5′-deoxy-5′-methylthioadenosine phosphorylase

Summary The distribution of 5-deoxy-5-methylthioadenosine (MTA) phosphorylase in 10 pig brain areas was determined. The observed regional differences of the enzymatic activity seem to reflect more the pattern of brain spermine distribution rather than that of spermidine. Moreover, comparative studies on the heat-resistance of MTA phosphorylase extracted from the whole brain of various species suggest structural differences in the enzyme molecules occurring in the brains of different animals.     

May K+ ions stimulate the formation of cyclic AMP in the brain independently on their depolarizing action?

Summary The effect of potassium ions on the formation of adenosine 3,5-monophosphate (cAMP) in the rat cerebral cortex in vivo was studied under conditions where development of spreading depression had been blocked by pretreatment of the cerebral cortex by topically applied magnesium ions. A linear relationship between potassium concentrations applied to the cortical surface and levels of cAMP has been found. Moreover, potentiation of the K⁺-effect by magnesium ions has been observed.     

Effects of several preoperative medications on fat cell lipolysis,and activity of adipose tissue cyclic AMP phosphodiesterase

Summary Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a clinical dose, omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 g ml^–1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compartible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.The authours are grateful to Dr D. C. Williams for his continued support and encouragement.     

Purification and some properties of an amine oxidase from soybean seedlings

Summary An amine oxidase was purified 447-fold from soybean seedlings and some of its properties were investigated. The molecular weight of the enzyme was estimated to be 25,000. It was most active towards putrescine, followed by spermidine and spermine. K_m-values for these substrates were relatively close. The enzyme was strongly inhibited by carbonyl reagents, such as semicarbazide and aminoguanidine.     

Inhibitory effects of spermine and spermidine on muscle calpain II

Summary The muscle enzyme calpain II, in contrast to muscle calpain I, was markedly inhibited by millimolar concentrations of the polyamines spermine and spermidine. These compounds and the calpain inhibitor calpastatin had synergistic inhibitory effects on calpain II. These results suggest that the polyamines may have possible regulatory effects on the in vivo activity of calpain II enzymes.     

Inhibitory effects of spermine and spermidine on muscle calpain II

The muscle enzyme calpain II, in contrast to muscle calpain I, was markedly inhibited by millimolar concentrations of the polyamines spermine and spermidine. These compounds and the calpain inhibitor calpastatin had synergistic inhibitory effects on calpain II. These results suggest that the polyamines may have possible regulatory effects on the in vivo activity of calpain II enzymes.     

Effect of dibutyryl cyclic AMP and analogs on the rate of contractions of myocytes in culture

Summary ²O,⁶N-butyryl, 3, 5-cyclic monophosphate (dibu cAMP) when added to fetal rat heart cells in culture inhibits myocyte contraction. This inhibition is 100, 84 and 51% complete when the dibu cAMP concentration used is 2, 0.2 and 0.02 mM, respectively. The potency of dibu cAMP derivatives in myocyte contraction inhibition follows the order, dibu cAMP> ⁶N-bu cAMP> ²O-bu cAMP=AMP>butyrate. The inhibition caused by the first three chemicals is greater than 70%.The author was a Moss Heart Fellow and acknowledges the technical assistance of Ms Martha Peet. This work was supported in part by the American Heart Association, National Science Foundation and American Cancer Society.     

Spermine protects protein kinase C from phospholipid-induced inactivation

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca²⁺. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca²⁺. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca²⁺ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca²⁺ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca²⁺ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation.     

Tannic acid inhibits insulin-stimulated lipogenesis in rat adipose tissue and insulin receptor function in vitro

Tannins occur naturally in relatively abundant amounts in fruits, herbal medicines and common beverages. Thus an understanding of how these polyphenols affect peptide hormone action is of importance. We report here that tannic acid (a hydrolysable tannin) inhibits insulin-stimulated lipogenesis in rat adipose tissue in vitro, with an IC₅₀ estimated to be about 350 M. However, its monomer, gallic acid, did not show a similar inhibitory effect at concentrations up to 1 mM. The inhibition by tannic acid was less evident with higher concentrations of bovine serum albumin in the incubation buffer. This was attributed to the formation of a tannin-protein complex between bovine serum albumin and tannic acid. In a binding assay, it was observed that the specific binding of insulin to its receptor was not inhibited by tannic acid in the concentration range 0–200 M. However, insulin-stimulated autophosphorylation of the insulin receptor, and receptor-associated tyrosine kinase phosphorylation of RR-SRC peptide, were inhibited by tannic acid at concentrations as low as 25 M. Our data do not support the current speculation that tannins affect the activity of peptide hormones by binding to them. Therefore, our finding opens up a new perspective in the understanding of the mode of action of tannins on such hormones.     

17β-Estradiol-sensitivity of cultured myometrial cells

Summary The estrogen sensitivity of cells cultured from the rat myometrium was studied by growing the cells in the absence or presence of 1 nM 17-estradiol. Following a time lag of 10 days, exposure to estrogen resulted in increased incorporation of radiothymidine by the cells. Estrogen treatment also decreased isoproterenol-dependent and GTP-dependent adenylate cyclase activity, but had no effect on basal activity. These cultured cells have been shown previously to have some properties of uterine smooth muscle. The effects estrogen has in viyro, therefore, may reflect important properties in vivo that account for the mechanism by which the sex steroid decreases the sensitivity of the myometrium to isoproterenol.     

Metallothionein induction by cadmium and zinc in rat secretory organs

Summary Metallothionein (MT) levels were determined in four secretory organs of the rat following administration of zinc (Zn) and cadmium (Cd). The concentrations of MT in the lacrimal, parotid and adrenal glands of untreated rats were in the range of 2.2–4.9 g/g wet weight tissue while in the pancreas it was shown to be 15.2 g/g. Injection of zinc at total doses of 16, 32 and 80 mg/kg resulted in a 1.8-, 3.2- and 5.9-fold increase in lacrimal MT content, respectively, while a 10.2- and 13.1-fold elevation was observed following treatment with 4 and 8 mg/kg of Cd, respectively. Similar findings were found in the adrenal gland. The parotid MT was elevated 5.9 and 17 times following Zn treatment at doses of 16 and 80 mg/kg respectively, whereas 4 mg/kg of Cd increased MT 14.4 times in this gland. Pancreatic MT was elevated by 39- and 40-fold after injection of Zn at doses of 16 and 32 mg/kg respectively, whereas 4 and 8 mg/kg of Cd caused a 9.8- and 17.9-fold induction, respectively. These results may indicate that secretory organs participate in metabolism of heavy metals in the mammalian body.     

Differential effects of prostaglandins and isoproterenol on cAMP content and Na,K pump activity in rat submandibular acini

Summary The -adrenergic agonist isoproterenol and prostaglandins E₁ and E₂ (but not F₂) increased the cAMP content of rat submandibular acini in vitro, but only isoproterenol enhanced ouabain-sensitive⁸⁶Rb (K) uptake. These findings suggest that cAMP is not involved in the activation of the Na, K pump in salivary cells.     

Effects of puromycin on rat embryos in vitro

Summary Somite-staged rat embryos were exposed to varying concentrations of puromycin for 48 h in vitro. Medium concentrations below 0.92 M had no significant effects, while concentrations above 1.84 M were lethal. Between these extremes, there were concentration dependent increases in the incidence of malformations in a close relationship to growth retardation.     

Purification and properties of ornithine aminotransferase from rat brain

Summary Ornithine aminotransferase (E.C. 2.6.1.13) from rat brain was purified 100-fold by ammonium sulphate fractionation, DEAE cellulose chromatography, calcium phosphate gel and alumina C gel. Pyridoxal phosphate was essential for maximum activity of the enzyme. The brain enzyme did not differ from liver and kidney enzymes in properties such as pH optimum, K_m, substrate specificity and the inhibition by branched chain amino acids. Unlike rat liver enzyme, brain ornithine aminotransferase was able to catalyze the reaction between L-lysine and 2-oxoglutarate. Spermidine and spermine inhibited brain ornithine aminotransferase activity.Acknowledgments. D.R.D. is thankful to U.G.C., India, for the award of a fellowship under the special assistance programme. Present address: Department of Pediatrics and Communicable Diseases, F2815, Box 066, C.S. Mott Children's Hospital, University of Michigan, Ann Arbor, MI 48109, USA.