Analysis of gene expression in mouse 2-cell embryos using fluorescein differential display: comparison of culture environments.
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The effect of the oviductal environment on gene expression in 2-cell mouse embryos was examined with mRNA differential display. Embryos used for experiments were cultured in modified Whitten medium with or without oviductal tissue until late 2-cell stage. The results of sequencing indicated that the genes for ATP synthase (ATPase 6), S:-adenosylmethionine decarboxylase (S:-AMDC) and nuclear autoantigenic sperm protein (NASP) were differentially expressed in embryos cultured in the oviductal environment (nonblocking culture condition). The ATPase 6 gene is encoded by mitochondrial DNA and is essential for the production of ATP. This indicates that the expression of ATP synthesis-related genes at the 2-cell stage may be required to maintain normal development in vitro. S:-Adenosylmethionine decarboxylase decarboxylates adenosylmethionine, which is a substrate of DNA methylation. The expression of S:-AMDC may be responsible for the low level of methylation of preimplantation development. As NASP is a histone-binding protein that is thought to be testis and sperm specific, its function in embryos remains unclear. On the other hand, the Tcl1 gene and a novel gene, the c-1 gene, were strongly expressed in embryos cultured without oviductal tissue (blocking culture condition). The expression patterns of these genes are quite similar. However, the detailed functions of these genes in embryos remain to be determined. (+info)
Characterization of monocot and dicot plant S-adenosyl-l-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames.
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S-Adenosyl-L-methionine decarboxylase (AdoMetDC; EC 4.1.1.50) is one of the key regulatory enzymes in the biosynthesis of polyamines. Isolation of genomic and cDNA sequences from rice and Arabidopsis had indicated that this enzyme is encoded by a small multigene family in monocot and dicot plants. Analysis of rice, maize and Arabidopsis AdoMetDC cDNA species revealed that the monocot enzyme possesses an extended C-terminus relative to dicot and human enzymes. Interestingly, we discovered that all expressed plant AdoMetDC mRNA 5' leader sequences contain a highly conserved pair of overlapping upstream open reading frames (uORFs) that overlap by one base. The 5' tiny uORF consists of two or three codons and the 3' small uORF encodes 50-54 residues. Sequences of the small uORFs are highly conserved between monocot, dicot and gymnosperm AdoMetDC mRNA species and the C-terminus of the plant small uORFs is conserved with the C-terminus of nematode AdoMetDC uORFs; such a conserved arrangement is strongly suggestive of a translational regulatory mechanism. No introns were found in the main AdoMetDC proenzyme ORF from any of the plant genes encoding AdoMetDC, whereas introns were found in conserved positions flanking the overlapping uORFs. The absence of the furthest 3' intron from the Arabidopsis gene encoding AdoMetDC2 suggests that this intron was lost recently. Reverse-transcriptase-mediated PCR analysis of the two Arabidopsis genes for AdoMetDC indicated that AdoMetDC1 is abundant and ubiquitous, whereas the gene for AdoMetDC2 is expressed preferentially in leaves and inflorescences. Investigation of recently released Arabidopsis genome sequences has revealed that in addition to the two genes encoding AdoMetDC isolated as part of the present work, four additional genes are present in Arabidopsis but they are probably not expressed. (+info)
Spermine deficiency resulting from targeted disruption of the spermine synthase gene in embryonic stem cells leads to enhanced sensitivity to antiproliferative drugs.
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Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts. (+info)
Antizyme regulates the degradation of ornithine decarboxylase in fission yeast Schizosaccharomyces pombe. Study in the spe2 knockout strains.
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The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe. (+info)
Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers.
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The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants. (+info)
A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast.
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In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity. (+info)
The story of CGP 40 215: studies on its efficacy and pharmacokinetics in African green monkey infected with Trypanosoma brucei rhodesiense.
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CGP 40 215 is an inhibitor of S-adenosylmethionine decarboxylase, a key enzyme in trypanosomal polyamine biosynthesis. It is highly active against Trypanosoma brucei rhodesiense and T. b. gambiense in vitro and in the corresponding rodent models, and therefore was a promising candidate for further development as a new drug against human African trypanosomiasis. We conducted initial pharmacokinetic and efficacy studies in African green monkeys: based on two dose-finding studies, an infection-treatment and a pharmacokinetic study in eight monkeys infected with T. b. rhodesiense in the 1st stage of infection. PK analysis revealed curative drug levels in the serum but complete absence of the drug in the cerebrospinal fluid. No adverse effects of the drug were observed, although in rats CGP 40 215 had caused hypotension. The following PK parameters were calculated using a two-compartment model: t1/2=1.8 h, VSS/f=0.4 l/kg, CL/f=3.0 ml/min x kg and AUC=21 900 ng x h/ml. Six of the eight monkeys were cured, one animal relapsed on day 222 and one animal died of unknown reasons, but was aparasitaemic. The study confirmed the curative potential of CGP 40 215 for 1st stage T. b. rhodesiense infection. Unfortunately, it was also found that the compound did not pass the blood-brain barrier, a pre-requisite for cure of 2nd stage (CNS) infection. As the majority of sleeping sickness patients seeking treatment are in the 2nd stage of the disease, further development of the compound was stopped. (+info)
The Plasmodium falciparum bifunctional ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, enables a well balanced polyamine synthesis without domain-domain interaction.
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In the human malaria parasite Plasmodium falciparum (Pf), polyamines are synthesized by a bifunctional enzyme that possesses both ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase (AdoMetDC) activities. The mature enzyme consists of the heterotetrameric N-terminal AdoMetDC and the C-terminal dimeric ODC, which results in the formation of a heterotetrameric complex. For the native bifunctional protein a half-life longer than 2 h was determined, which is in contrast to the extreme short half-life of its mammalian monofunctional counterparts. The biological advantage of the plasmodial bifunctional ODC/AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein. An interesting feature in the regulation of the bifunctional protein is that putrescine inhibits PfODC activity approximately 10-fold more efficiently than the mammalian ODC activity, and in contrast to the mammalian AdoMetDC the activity of the PfAdoMetDC domain is not stimulated by the diamine. To analyze post-translational processing, polymerization, and domain-domain interactions, several mutant proteins were generated that have single mutations in either the PfODC or PfAdoMetDC domains. The exchange of amino acids essential for the activity of one domain had no effect on the enzyme activity of the other domain. Even prevention of the post-translational cleavage of the AdoMetDC domain or ODC dimerization and thus the interference with the folding of the protein hardly affected the activity of the partner domain. In addition, inhibition of the activity of the PfODC domain had no effect on the activity of the PfAdoMetDC domain and vice versa. These results demonstrate that no domain-domain interactions occur between the two enzymes of the bifunctional PfODC/AdoMetDC and that both enzymatic activities are operating as independent catalytic sites that do not affect each other. (+info)