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Treatment of human T lymphocytes with mitogenic ligands, such as concanavalin A (Con A), induces a rapid activation of the enzyme ornithine decarboxylase (ODC). This activation occurs within minutes and is completely inhibited when the cells are treated with 1 mM Li+ (in an inositol-free medium) prior to stimulation with Con A. In the presence of 1 mM myo-inositol Li+ has no effect on the Con A-induced activation of ODC. To elucidate why inositol is needed for the mitogen-induced activation of ODC in T lymphocytes, we tested the ability of different inositol metabolites to reverse the inhibitory effect of Li+. Here we report that inositol phospholipids, in addition to inositol, reverse the Li+-induced inhibition of ODC activation, while all other inositol derivatives tested were ineffective. This indicates that Li+ does not block the activation of ODC by inhibiting the generation of inositol phosphates, but rather by a mechanism which is circumvented if inositol phospholipids are added. The molecular mechanisms involved in the rapid activation of ODC by mitogens in human T lymphocytes apparently require inositol phospholipids, but are not directly mediated by inositol-1,4,5-trisphosphate (IP3) alone, diacylglycerol alone, or other inositol phosphates.

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We have previously shown that treatment of T lymphocytes with mitogenic ligands induces a rapid activation of ornithine decarboxylase (ODC) through a mechanism that is independent of protein synthesis but requires energy and an intact cytoskeleton. Here we show by immunoprecipitation experiments and by chemical analyses that ODC is covalently linked to the cell membrane by inositol. Treatment of sonicated cells with a phosphatidylinositol-specific phospholipase C from B. thuringiensis caused a rapid 3-fold increase in ODC activity. Similar treatment of intact cells had no effect, suggesting that the ODC is attached to the cytoplasmic surface of the membrane. We conclude that ODC release and activation occur by a novel mechanism involving phosphatidylinositol breakdown following ligand-receptor interaction.

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Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts of Mycobacterium bovis (BCG). All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg2+-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria.

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Rapid activation of ornithine decarboxylase is one of the earliest recognized events during induction of a mitogenic response in human T lymphocytes. Here we show that the non-hydrolysable GTP analogues guanine-5-(gamma-thio)trisphosphate and guanylyl-5-imidodiphosphate, introduced into human T cells by means of a transient membrane permeabilization technique, can replace an external mitogenic ligand, such as concanavalin A, in inducing early ornithine decarboxylase activity. Neomycin inhibits this rapid activation at concentrations known to bind to phosphoinositides. One of the two compounds formed in polyphosphoinositide breakdown, inositol-1,4,5-trisphosphate, also induces ornithine decarboxylase activity. The other, diacylglycerol, apparently does not, since the phorbol ester, tetradecanoyl phorbol acetate, which is thought to mimic the action of diacylglycerols, does not alter basal ornithine decarboxylase activity in T cells until several hours after administration. These findings indicate that guanine nucleotide-binding regulatory (G-) protein(s) participates in the transduction of the mitogenic signal. The intracellular target system for this G-protein may include phosphoinositide breakdown and generation of inositoltrisphosphate, which might be involved in the early activation of ornithine decarboxylase.

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One of the earliest detectable responses by T lymphocytes in vitro to various mitogenic ligands is the rapid induction of ornithine decarboxylase (ODC) activity (Scott et al., Eur. J. Immunol. 1985. 15: 783). This early activation is measurable within minutes after the addition of the mitogen. The T cell mitogen concanavalin A also induces rapid breakdown of phosphoinositides in T lymphocytes. The inositol phosphates formed are recycled and used in synthesis of new phosphoinositides. Li+ interrupts this cycle by inhibiting the enzyme inositol-1-phosphatase, which produces the inositol needed for phosphoinositide synthesis. Here we report that when human blood lymphocytes are kept in an inositol-deficient medium for 30 min in the presence of 1 mM Li+, the cells become unable to respond to mitogens by inositide breakdown and rapid induction of ODC activity. Addition of 1 mM myo-inositol restores the inducibility of these responses within a few minutes. These findings represent a novel aspect of the activation of resting lymphocytes and implies a new role for the inositol turnover during signal transduction.

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Cadaverine links covalently to the D-glutamic acid residue of the peptidoglycan in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium (Kamio, Y., Itoh, Y., and Terawaki, Y. (1981) J. Bacteriol. 146, 49-53). This report clarifies a physiological function of cadaverine in this organism by using DL-alpha-difluoromethyllysine, which had previously been shown to be a selective irreversible inhibitor of lysine decarboxylase of Mycoplasma dispar (Pösö, H., MaCann, P.P., Tanskanen, R., Bey, P., and Sjoerdsma, A. (1984) Biochem. Biophys. Res. Commun. 125, 205-210). DL-alpha-Difluoromethyllysine is now shown to be a potent and irreversible inhibitor of lysine decarboxylase of S. ruminantium in vitro; however, it did not inhibit the transfer of cadaverine to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan. DL-alpha-Difluoromethyllysine at 5 mM markedly inhibited the growth of the bacterium and caused rapid cell lysis. Immediately before the cell lysis, almost all cells became swollen, and such cells showed a loosened envelope structure when studied by electron microscopy. The peptidoglycan prepared from the DL-alpha-difluoromethyllysine-treated cells did not have covalently linked cadaverine. The growth inhibition by DL-alpha-difluoromethyllysine was completely reversed by adding cadaverine (1 mM) to the medium. Furthermore, the exogenous cadaverine was exclusively incorporated into the peptidoglycan in the presence of DL-alpha-difluoromethyllysine (5 mM), and a normal peptidoglycan was synthesized. The cell lysis and the formation of an abnormal cell structure were completely prevented by cadaverine added to the medium. We conclude that cadaverine covalently linked to the peptidoglycan in S. ruminantium is an essential constituent of the peptidoglycan and is required for cell surface integrity and the normal growth of S. ruminantium.

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Dicyclohexylamine, which is an inhibitor of bacterial and mammalian spermidine synthase, greatly inhibited the synthesis of spermidine in Pseudomonas aeruginosa. The depletion of spermidine caused by dicyclohexylamine was accompanied by an inhibition of growth of bacteria. This inhibition was reversed by addition of 50 microM spermidine (but not putrescine or spermine) to growth medium. When its growth was inhibited Ps. aeruginosa also lost its motility. Electron microscopy showed a loss of flagella in spermidine-deficient bacteria: after 24 h 70% 85% of bacteria grown in the presence of dicyclohexylamine did not have flagella, whereas bacteria grown in the presence of dicyclohexylamine and spermidine had flagella. The loss of flagella was reversible, since after the inhibition of spermidine synthesis for 24 h, addition of 50 microM spermidine (but not putrescine or spermine) to the growth medium was able to restore the bacterial motility almost completely after a further 12 h growth period.

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The putrescine-stimulated S-adenosyl-L-methionine decarboxylases from rat liver and yeast were strongly inhibited by Berenil and to a lesser extent by Pentamidine. Ten times greater drug concentrations were needed to achieve a similar level of inhibition of a Mg2+-stimulated bacterial enzyme. The inhibition was irreversible in that extensive dialyses or precipitation with (NH4)2SO4 did not restore enzyme activity. Putrescine did not protect the enzyme against Berenil, but adenosylmethionine either alone or with putrescine partially protected the irreversible action of Berenil. The compound 4,4'-diamidinodiphenylamine, which differs from Berenil only in lacking the azo group between benzene rings, was a weaker inhibitor than Berenil, and its inhibition was reversible. Berenil also inhibited the activity of adenosylmethionine decarboxylase in vivo, by depressing the activity of the enzyme in normal rat liver, for at least 24 h after a single injection (50 mg/kg body wt.) of the drug.

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The activity of coumarin 7-hydroxylase (coumarin 7-hydroxylation) was inhibited in B6 mouse liver after a single injection of methylglyoxal bis(guanylhydrazone (MGBG). The decrease in the activity in vivo was greatest (70%) one day after the drug injection and the hydroxylase activity in microsomal fraction prepared from livers of MGBG-treated B6 mice was still 25% decreased 5 days after the drug. The amount of cytochrome P-450 also was decreased in MGBG-treated livers with the same time-dependency as the inhibition of coumarin 7-hydroxylation. MGBG and its close derivative 1,1'-[methylethanediylidene)dinitrilo)bis(3-aminoguanidine) (MBAG) inhibited the activity in vitro of coumarin 7-hydroxylase, benzo(a)pyrene hydroxylase and 7-ethoxy 0-de-ethylase when microsomes were prepared from livers of untreated B6 mice. In every case MBAG was a better inhibitor than MGBG in vitro. The in vitro inhibition of MGBG of several drug metabolizing enzymes was not reversed when microsomes were preincubated with 1 mM putrescine, spermidine or spermine. These results suggest that the anti-cancer drug, MGBG, has a severe effect(s) on the drug metabolizing system at concentrations reached during the treatment of patients with MGBG.

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The T cell mitogens, concanavalin A and the monoclonal antibody OKT3, cause a rapid activation of ornithine decarboxylase (ODC) activity in human T lymphocytes, maximal within 10 min of mitogen addition. Nonmitogenic ligands to T cell surface structures do not induce ODC. The enzyme induction is dependent upon an intact mobility of ligand-receptor complex, requires a functioning energy metabolism, but is independent of de novo protein synthesis. The early induction of pre-existing ODC molecules appears to be specifically linked to the initiation of T lymphocyte proliferation.

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Only the dextro isomer of ethambutol inhibited mycobacterial spermidine synthase and spermidine biosynthesis. Inhibition of mycobacterial spermidine synthase appeared to be specific. Spermidine synthase from Mycobacterium fortuitum, which was resistant to ethambutol in vitro, required a higher concentration of ethambutol for its inhibition than the enzyme of susceptible Mycobacterium bovis.

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1-Aminooxy-3-aminopropane was shown to be a potent competitive inhibitor (Ki = 3.2 nM) of homogenous mouse kidney ornithine decarboxylase, a potent irreversible inhibitor (Ki = 50 microM) of homogeneous liver adenosylmethionine decarboxylase and a potent competitive (Ki = 2.3 microM) of homogeneous bovine brain spermidine synthase. It did not inhibit homogeneous bovine brain spermine synthase and it did not serve as a substrate for spermidine synthase. The compound did not inhibit tyrosine aminotransferase, alanine aminotransferase or aspartate aminotransferase, which are pyridoxal phosphate-containing enzymes like ornithine decarboxylase. The inactivation of adenosylmethionine decarboxylase was partially prevented by pyruvate, which is the coenzyme of adenosylmethionine decarboxylase, and by the substrate, adenosylmethionine. 1-Aminooxy-3-aminopropane at 0.5 mM concentration inhibited the growth of HL-60 promyelocytic leukemia cells and this inhibition was prevented by spermidine but not by putrescine.

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Physiological concentrations (0.5-2.0 mM) of spermidine and spermine were observed to inhibit the digestion in vitro of plasmid pJDB 207 by the restriction endonucleases BamHI (EC 3.1.23.6), EcoRI (EC 3.2.23.13), HindIII (EC 3.1.23.20), HpaI (EC 3.1.23.23) and PstI (EC 3.1.23.31). The polyamines protected all the tested restriction sequences of DNA, since the activity of all endonucleases used was strongly inhibited. These results show the need for caution when using polyamines as experimental tools for recombinant DNA chemistry.

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A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.

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The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.

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The effect of dicyclohexylamine on seven freshly isolated bacterial strains of mastitis pathogens was studied. Streptococcus uberis was the most sensitive strain investigated, since 5 mM-dicyclohexylamine totally arrested its growth and 1.25 mM of the drug caused 60% growth inhibition. The Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains were also sensitive to the drug, but less so than Strep. uberis, since 5 mM drug caused only partial inhibition of growth. Micrococcus sp. and Klebsiella sp. grew in the presence of 10.0 mM-dicyclohexylamine, and, finally the growth of Streptococcus agalactiae was not at all affected by dicyclohexylamine. These different sensitivities towards dicyclohexylamine in vivo were paralleled by different sensitivities of the bacteria's spermidine synthase to the drug in vitro, and also by the ability of the drug to lower spermidine concentration in bacterial cells. Spermidine synthase from sensitive bacteria was inhibited by more than 90% by 50 microM-dicyclohexylamine in vitro, and the concentration of spermidine was decreased in E. coli and Ps. aeruginosa by 70% and in Strep. uberis by 95%, whereas in Strep. agalactiae 5 mM-dicyclohexylamine did not affect the concentration of spermidine at all. Dicyclohexylamine treatment led to the accumulation of putrescine in Strep. uberis. Spermidine synthesis catalysed by the extracts of Micrococcus sp. required 500 microM-dicyclohexylamine for 90% inhibition, and Strep. agalactiae contained a spermidine synthase that was still active at 1000 microM-dicyclohexylamine, The observed inhibition of growth was totally reversed by adding 50 microM-spermidine (final concentration) to the medium. Putrescine reversed the inhibition only when bacteria had a spermidine synthase activity insensitive to dicyclohexylamine. Spermine did not overcome the inhibition of growth caused by dicyclohexylamine, probably because it was not taken up by the bacterial cells used in this study. The inhibition of the growth by dicyclohexylamine (even in the case of Strep. uberis) was reversible in the sense that addition of 50 microM-spermidine 18 h after dicyclohexylamine still restored the growth rate of untreated controls.

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DL-alpha-Difluoromethyllysine was shown to be a potent inhibitor of lysine decarboxylase from Mycoplasma dispar. The inhibition appeared to be specific since neither difluoromethylornithine nor difluoromethylarginine, known to inhibit other decarboxylases, inhibited the reaction catalyzed by lysine decarboxylase in extracts of M. dispar. Inhibition was irreversible since extensive dialysis could not overcome the inhibitory effect exerted by difluoromethyllysine. Difluoromethyllysine (1 mM) also totally blocked the growth of M. dispar when added at the beginning of the growth period, while 1 mM cadaverine, the product of the reaction, reversed this inhibitory effect when added to the culture medium. When difluoromethyllysine was added during the logarithmic growth phase of Mycoplasma, it inhibited the increase of the growth; 1 mM cadaverine again partially reversed this inhibitory action.

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A human neuroblastoma cell line (Paju) was resistant to 10 mM difluoromethylornithine, a concentration at which the growth of all mammalian cells normally stops. Ornithine decarboxylase from Paju was very resistant to inhibition by difluoromethylornithine in vitro (Ki = 10 microM compared to 0.5 microM for mouse kidney ornithine decarboxylase). After purification, apparently homogeneous Paju ornithine decarboxylase was inactivated with [3H]difluoromethylornithine and analyzed by polyacrylamide gel electrophoresis. Under denaturing conditions it was found to have an altered molecular structure, i.e. two nonidentical subunits of Mr = 55,000 and 60,000. Another unusual feature of Paju ornithine decarboxylase was its long half-life in vivo (T 1/2 = 8 h compared with 36 min in human HL-60 promyelocytic leukemia cells). The disappearance of immunoreactive protein was only slightly slower than the loss of catalytic activity. The long half-life of Paju ornithine decarboxylase was not shared by adenosylmethionine decarboxylase. Despite the altered structure of Paju ornithine decarboxylase, it was recognized by a specific antisera raised in rabbit against mouse kidney ornithine decarboxylase. The Paju karyotype did not contain double minute chromosomes or any large homogeneously staining region such as that seen in a mouse lymphoma cell mutant that is resistant to difluoromethylornithine and overproduces ornithine decarboxylase (McConlogue, L., and Coffino, P. (1983) J. Biol. Chem. 258, 12083-12086).

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When the ability of T4-DNA ligase from E. coli NM 989 to form higher molecular weight polymers from linearized plasmid pJDB 207 was followed, it was observed that physiological concentrations (0.5 to 1.0 mM) of spermidine and spermine greatly stimulated the formation of these polymers. The effect had a strict specificity since 1,3-diaminopropane, putrescine (1,4-diaminobutane) and N1-acetylspermidine neither stimulated nor inhibited this activity of DNA ligase. The structural analogues of spermidine, methyl bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-aminoguanidine) totally abolished the stimulatory effect of spermidine on T4-DNA ligase without affecting the enzyme's basal activity.

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