Addition Of Exogenous Spermidine Research Articles

Insight Into Genes Involved in the Production of Extracellular Chitinase in a Biocontrol Bacterium Lysobacter enzymogenes C-3

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.

Spermidine is essential for normal proliferation of trypanosomatid protozoa

Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of α-difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis-cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.

Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression

Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G 2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine- or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G 1 to S phase transition.

Treatment of nude mice with 4-amidinoindan-1-one2???-amidinohydrazone, a new S-adenosylmethionine decarboxylase inhibitor, delays growth and inhibits metastasis of human melanoma cells

CGP 48664A (4-amidinoindan-1-one2'-amidinohydrazone) is a novel inhibitor of S-adenosyl-methionine decarboxylase (SAMDC), a key enzyme in the biosynthesis of polyamines, which are themselves essential for proliferation of mammalian cells. Seven different human melanoma cell lines were treated in vitro with CGP 48664A. High, intermediate and low levels of cytostasis were induced in four, one and two melanoma lines, respectively. This cytostasis was reversed by the addition of exogenous spermidine or spermine to the culture medium. The heterogeneous low metastatic (CGP 48664A-resistant) A375P cells and highly metastatic (CGP 48664A-sensitive) A375SM cells were implanted into the subcutis or injected intravenously into nude mice. Systemic daily administration of CGP 48664A significantly reduced the size of cutaneous lesions and the number of lung metastases in mice implanted with A375SM cells. No beneficial effects were found in mice injected with A375P cells. Drug activity was dose dependent, and maximal effects were observed when treatment began in mice with small tumour burdens. The data suggest that CGP 48664A is effective against melanoma metastasis in nude mice and that its activity should be tested in combination with other cytoreductive agents.

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (α-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (α-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.

Inhibition of S-Adenosyl-L-Methionine (AdoMet) Decarboxylase by the Decarboxylated AdoMet Analog 5'-[(Z)-4-Amino-2-Butenyl]Methylamino-5'-Deoxyadenosine (MDL 73811) Decreases the Capacities of Trypanosoma cruzi to Infect and Multiply within a Mammalian Host Cell

A decarboxylated S-adenosyl-L-methionine (AdoMet) analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811), that specifically and irreversibly inhibits AdoMet decarboxylase (DC) was used to investigate the role of AdoMetDC in the regulation of host cell invasion and intracellular replication by Trypanosoma cruzi. The presence of MDL 73811 in cocultures of T. cruzi and rat heart myoblasts (RHM) significantly inhibited host cell infection in a dose-dependent manner. This effect, evidenced by reductions in the proportion of infected RHM and the number of parasites per 100 RHM, was due to MDL 73811 action on T. cruzi as it was reproduced when the parasites, but not the RHM, were pretreated with the inhibitor. Significant inhibition of infectivity required a 10-min treatment with MDL 73811 although greater effects ensued with additional incubation time. We could not detect signs of recovered infectivity up to 6 hr after the removal of nonincorporated MDL 73811, suggesting that either AdoMetDC turnover in T. cruzi is a relatively slow process or the amounts of MDL 73811 taken up could not be blocked or eliminated during this time period. MDL 73811-mediated inhibition of infectivity was not bypassed by the addition of exogenous spermidine or spermine, suggesting that the mechanism of action does not involve reduced production of these polyamines due to AdoMetDC inhibition. Intracellular accumulation of AdoMet appeared to be a more plausible explanation in view of the fact that exogenous AdoMet significantly inhibited infectivity. When added to infected RHM cultures, MDL 73811 or AdoMet inhibited also intracellular T. cruzi growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chlorsulfuron on polyamine content in maize seedlings

The effects of chlorsulfuron, a herbicide known to inhibit plant growth by inhibiting cell cycle progression in both G 1 and G 2 stages, were examined on free polyamine content and on growth in Zea mays seedlings. Chlorsulfuron decreased the spermidine content in active growing tissue, such as root tips, completely unaffecting the spermidine content in the basal part of the root where mitotic activity is lacking. In addition to the effects of specific spermidine inhibitors, methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase activity, cyclohexylamine, a competitive inhibitor of spermidine synthase activity, and chlorsulfuron were examined on both free and bound polyamines. Chlorsulfuron proved to be able to decrease the free and bound spermidine content more than methylglyoxal-bis(guanylhydrazone) and cyclohexylamine but probably through a different mechanism. In fact the inhibition of free spermidine content induced by methylglyoxal-bis(guanylhydrazone) and cyclohexylamine was reversed by the addition of exogenous spermidine, while no reversal effect was obtained for the inhibition of spermidine content induced by chlorsulfuron. It has been hypothesized that the depletion of spermidine content, induced by chlorsulfuron, could be responsible for the inhibition of cell cycle progression in G 1 and G 2 stages leading to growth inhibition.

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60.

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.

Dicyclohexylamine effects on HTC cell polyamine content and ornithine decarboxylase activity

Dicyclohexylamine, a spermidine synthase inhibitor, was evaluated for its ability to alter specific polyamine levels in rat hepatoma HTC cells in culture. Media concentrations of 0.5 and 1.0 mM reduced the production of spermidine from putrescine and enhanced the conversion of existing spermidine to spermine. This created a very interesting change in polyamine levels such that after 24 h putrescine content was almost 3-times control values and spermine was about twice, while spermidine was lowered to about 10% of control cultures. This pattern of polyamines is quite distinct from that induced by the common polyamine biosynthetic inhibitors like methylglyoxal bis(guanylhydrazone) and difluoromethylornithine and replicates the pattern induced by S-adenosyl-1,8-diamino-3-thiooctane, a transition-state analog designed as a specific inhibitor of spermidine synthase. When cells were stimulated by serum addition, the presence of dicyclohexylamine caused an extraordinarily large induction in ornithine decarboxylase in spite of the abnormally high levels of both putrescine and spermine. The concomitant depression of spermidine levels induced a 4-fold increase in the stability of this enzyme that could be reversed by the addition of exogenous spermidine. The data suggest that spermidine induces, perhaps at the transcriptional level, a protein that is necessary for the characteristically very rapid inactivation of ornithine decarboxylase.