BACKGROUND

Functional probiotics may prevent Helicobacter pylori infection, and some evidence suggests that they also possess antitumor properties. Lactobacillus brevis (CD2) is a functional Lactobacillus strain with peculiar biochemical features, essentially related to the activity of arginine deiminase. This enzyme catalyzes the catabolism of arginine and affects the biosynthesis of polyamines (putrescine, spermidine, and spermine). Polyamines are polycations found in high concentrations in both normal and neoplastic cells. Our aims were: 1, to assess whether oral administration of L. brevis (CD2) affects H. pylori survival in the human gastric mucosa; 2, to evaluate the effects of L. brevis (CD2) on polyamine biosynthesis in gastric biopsies from H. pylori-positive patients.

METHODS

For 3 weeks before endoscopy, 22 H. pylori-positive dyspeptic patients randomly received (ratio 1 : 1) high oral doses of L. brevis (CD2) or placebo. Before and after treatment, H. pylori infection was determined by urea breath test (UBT). In gastric biopsies, ornithine decarboxylase activity and polyamine levels were, respectively, evaluated by a radiometric technique and high-pressure liquid chromatography (HPLC).

RESULTS

L. brevis (CD2) treatment did not eradicate H. pylori. However, a reduction in the UBT delta values occurred, suggesting a decrease in intragastric bacterial load. Significantly, L. brevis (CD2) induced a decrease in gastric ornithine decarboxylase activity and polyamine levels.

CONCLUSIONS

Our data support the hypothesis that L. brevis (CD2) treatment decreases H. pylori colonization, thus reducing polyamine biosynthesis. Alternatively, the arginine deiminase activity following L. brevis (CD2) administration might cause arginine deficiency, preventing polyamine generation from gastric cells.

Spermine oxidase (SMO) was discovered much more recently than other enzymes involved in polyamine metabolism; this review summarizes 10 years of researches on this enzyme. Spermine oxidase (SMO) is a FAD-dependent enzyme that specifically oxidizes spermine (Spm) and plays a dominant role in the highly regulated mammalian polyamines catabolism. SMO participates in drug response, apoptosis, response to stressful stimuli and etiology of several pathological conditions, including cancer. SMO is a highly inducible enzyme, its deregulation can alter polyamine homeostasis, and dysregulation of polyamine catabolism is often associated with several disease states. The oxidative products of SMO activity are spermidine, and the reactive oxygen species H(2)O(2) and the aldehyde 3-aminopropanal each with the potential to produce cellular damages and pathologies. The SMO substrate Spm is a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function, cellular signaling, nitric oxide synthesis and inhibition of immune responses. The goal of this review is to cover the main biochemical, cellular and physiological processes in which SMO is involved.

Autophagy is a cell self-digestion process via lysosomes that clears "cellular waste", including aberrantly modified proteins or protein aggregates and damaged organelles. Therefore, autophagy is considered a protein and organelle quality control mechanism that maintains normal cellular homeostasis. Dysfunctional autophagy has been observed in ageing tissues and several ageing-associated diseases. Lifespan of model organisms such as yeast, worms, flies, and mice can be extended through promoting autophagy, either by genetic manipulations such as over-expression of Sirtuin 1, or by administrations of rapamycin, resveratrol or spermidine. The evidence supports that autophagy may play an important role in delaying ageing or extending lifespan. In this review, we summarize the current knowledge about autophagy and its regulation, outline recent developments ie the genetic and pharmacological manipulations of autophagy that affects the lifespan, and discuss the role of autophagy in the ageing-related diseases.

Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.

Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

To investigate possible biochemical mechanisms underlying the "toxic gain of function" associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-M(r) aggregates--a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral-pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptides in vitro, resulting in the formation of high-M(r) aggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60 polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-M(r) protein aggregates, thereby contributing to cell death.

Activation of polyamine catabolism through the overexpression of spermidine/spermine N1-acetyltransferase (SSAT) in transgenic rodents does not only lead to distorted tissue polyamine homeostasis, manifested as striking accumulation of putrescine, appearance N1-acetylspermidine and reduction of tissue spermidine and/or spermine pools, but likewise creates striking phenotypic changes. The latter include loss of hair, lipoatrophy and female infertility. Forced expression of SSAT modulates skin, prostate and intestinal carcinogenesis, induces acute pancreatitis and blocks early liver regeneration. Although many of these features are directly attributable to altered tissue polyamine pools, some of them are more likely related to the greatly accelerated flux of the polyamines caused by activated catabolism and compensatorily enhanced biosynthesis.

Human EBP1, isolated from HeLa cells, binds to a 10-base-pair (bp) sequence in cellular and viral enhancers that is also recognized by the inducible transcription factor NF-kappa B. Here we describe the interaction of purified EBP1 with the 10-bp repeated sequence that is responsive to signals which activate T cells and which form part of the human immunodeficiency virus type 1 (HIV-1) enhancer. DNase I footprinting indicates that both 10-bp sites on the same molecule, located between -80 and -105 on the HIV-1 long terminal repeat, can be occupied by EBP1, while dimethyl sulfate protection and methylation interference experiments indicate which purine bases are in contact with the protein. The presence of bases which exhibit increased rates of dimethyl sulfate-induced methylation in the presence of EBP1 indicate that interaction of EBP1 with its recognition site is accompanied by distortion of the DNA double helix. Supporting this conclusion is the observation that the polyamine spermidine dramatically increases EBP1 binding to its cognate site on the DNA. Studies with human T cells (Jurkat) and nucleotide stimulation data suggest that EBP1 is the activated form of NF-kappa B in these cells.

The cytotoxic response of the human large cell lung carcinoma line NCI H157 to exposure to the polyamine analogue N1 N12-bis(ethyl)spermine (BESpm) is preceded by an extremely high induction of spermidine/spermine N1-acetyltransferase (SSAT). The human enzyme has now been purified greater than 300-fold to apparent homogeneity and found to cross-react with antisera raised against rat liver SSAT. Although other acetylases are capable of acetylating polyamines using acetyl-CoA as the acetyl donor, the greater than 600-fold induction within 24 h was found to be specifically SSAT, since essentially all activity was precipitable by the specific antisera. The human enzyme appears to be similar to the rat enzyme in subunit size under reducing conditions (approximately 20 kDa), substrate specificity and kinetic parameters. Preliminary results using actinomycin D and cycloheximide suggested that the unusually high induction by N1 N12-bis(ethyl)spermine in the human lung cancer line result from new mRNA and protein synthesis. This hypothesis is further substantiated here by 'in vitro' translation experiments comparing poly(A) mRNA from control and treated cells. The large cell lung carcinoma line NCI H157 represents a useful system to produce large amounts of the SSAT protein and to study the molecular events responsible for the induction and control of this important polyamine-metabolic enzyme. By using this rich source of SSAT protein, a partial amino acid sequence was determined by N-terminal sequencing of endoproteinase Lys-C digestion fragments. Further, this system should be useful in determining whether there is an association between the unusually high induction of the acetylase and the observed cytotoxicity in the NCI H157 cells.

An activated Ki-ras was expressed in the human colon adenocarcinoma cell line Caco-2 to study the effects of Ki-ras oncogene on polyamine metabolism during gastrointestinal tumorigenesis. Multiple clones selected for expression of the mutant Ki-ras transgene displayed a suppression of transcription of a key catabolic enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Gene expression analysis, with cDNA microarrays, showed that Ki-ras transfected clones had decreased levels of expression, compared to mock transfected cells, of peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation. The activated Ki-ras suppressed SSAT expression by a mechanism involving the PPARgamma response element (PPRE) located at +48 bp relative to the transcription start site of the SSAT gene. Transient expression of the PPARgamma protein in Ki-ras expressing Caco-2 clones, or treatment with the PPARgamma ligand ciglitazone, led to an increase in the SSAT promoter activity. A MEK1/2 inhibitor PD98059 induced transcription of both PPARgamma and SSAT genes in the activated Ki-ras clones, suggesting that the mitogen-activated protein kinases (MAPKs) were involved in the regulation of SSAT expression by PPARgamma. We concluded that mutated Ki-ras suppressed SSAT via a transcriptional mechanism involving the PPARgamma signaling pathway.

A widespread increase in SpmS (spermine synthase) activity has been produced in transgenic mice using a construct in which the human SpmS cDNA was placed under the control of a composite CMV-IE (cytomegalovirus immediate early gene) enhancer-chicken beta-actin promoter. Four separate founder CAG/SpmS mice were studied. Transgenic expression of SpmS was found in all of the tissues examined, but the relative SpmS activities varied widely according to the founder animal and the tissue studied. Very large increases in SpmS activity were seen in many tissues. SpdS (spermidine synthase) activity was not affected. Although there was a statistically significant decline in spermidine content and increase in spermine, the alterations were small compared with the increase in SpmS activity. These results provide strong support for the concept that the levels of the higher polyamines spermidine and spermine are not determined only by the relative activities of the two aminopropyltransferases. Other factors such as availability of the aminopropyl donor substrate decarboxylated S-adenosylmethionine and possibly degradation or excretion must also influence the spermidine/spermine ratio. No deleterious effects of SpmS overexpression were seen. The mice had normal growth, fertility and behaviour up to the age of 12 months. However, breeding the CAG/SpmS mice with MHC (alpha-myosin heavy chain)/AdoMetDC (S-adenosylmethionine decarboxylase) mice, which have a large increase in S-adenosylmethionine decarboxylase expression in heart, was lethal. In contrast, breeding the CAG/SpmS mice with MHC/ODC (L-ornithine decarboxylase) mice, which have a large increase in cardiac ornithine decarboxylase expression, had a protective effect in preventing the small decrease in viability of the MHC/ODC mice.

We extend the scope of nanometer distance measurements based on coupled pairs of gold nanoparticles, or plasmon rulers, to individual RNA molecules. These sensors were used to monitor the influence of spermidine on the cleavage kinetics of RNA by ribonuclease A. Time-resolved cleavage experiments of individual RNA plasmon rulers reveal transiently stabilized RNA sub-populations at increased spermidine concentrations that indicate spermidine-induced stabilization of weak secondary and tertiary structural elements.

BACKGROUND

Many gram-negative bacterial pathogens employ Type III secretion systems (T3SS) to inject effector proteins into host cells in infectious processes.

RESULTS

By screening a transposon mutant library of P. aeruginosa, we found that mutation of spuDEFGH, which encode a major spermidine uptake system, abolished the expression of the exsCEBA operon that codes for key T3SS regulators under inducing conditions (low calcium). Whole genome microarray analysis revealed that inactivation of the spermidine uptake system significantly decreased the transcriptional expression of most, if not all, T3SS genes. Consistently, the spermidine uptake mutants showed decreased expression of the T3SS genes in responding to host cell extract and attenuated cytotoxicity. Furthermore, exogenous addition of spermidine to the wild type strain PAO1 enhanced the expression of exsCEBA and also the effector protein genes.

CONCLUSIONS

Cumulatively, these data have depicted a novel spermidine transporter-dependent signaling pathway, which appears to play an essential role in modulation of T3SS expression in P. aeruginosa.

Peptide bond synthesis was studied with native polyribosomes of E. coli. With the use of this system for transpeptidation, it was possible to show that a single K(+) activates the ribosome monomers of polyribosomes; that protonation of a single group (probably imidazole or an N-terminal amino group) with a pK(a) equal to about 7.2 inactivates the transpeptidase complex; that Mn(++) can substitute for Mg(++), but that Ca(++), spermidine, and putrescine do so only very poorly; and that the K(m) for puromycin in this system is about 2.4 x 10(-6) M.

DNA topoisomerase activity detected in cell extracts of the trypanosomatid Crithidia fasciculata interlocks kinetoplast DNA duplex minicircles into huge catenane forms resembling the natural kinetoplast DNA networks found in trypanosomes. Catenation of duplex DNA circles is reversible and equilibrium is affected by ionic strength, and by spermidine. The reaction requires magnesium, is ATP dependent and is inhibited by high concentrations of novobiocin. Extensive homology between duplex DNA rings was not required for catenane formation since DNA circles with unrelated sequences could be interlocked into mixed network forms. Covalently sealed catenaned DNA circles are specifically used as substrates for decatenation. No such preference for covalently sealed duplex DNA rings was observed for catenate formation. Its catalytic properties and DNA substrate preference, suggest a potential role for this eukaryotic topoisomerase activity in the replication of kinetoplast DNA.

We developed a system for DNA packaging of isolated bacteriophage T4 proheads in vitro and studied the role of prohead expansion in DNA packaging. Biologically active proheads have been purified from a number of packaging-deficient mutant extracts. The cleaved mature prohead is the active structural precursor for the DNA packaging reaction. Packaging of proheads requires ATP, Mg2+ and spermidine, and is stimulated by polyethylene glycol and dextran. Predominantly expanded proheads (ELPs) are produced at 37 degrees C and predominantly unexpanded proheads (ESPs) are produced at 20 degrees C. Both the expanded and unexpanded proheads are active in DNA packaging in vitro. This is based on the observations that (1) both ESPs and ELPs purified by chromatography on DEAE-Sephacel showed DNA packaging activity; (2) apparently homogeneous ELPs prepared by treatment with sodium dodecyl sulfate (which dissociates ESPs) retained significant biological activity; (3) specific precipitation of ELPs with anti-hoc immunoglobulin G resulted in loss of DNA packaging activity; and (4) ESPs upon expansion in vitro to ELPs retained packaging activity. Therefore, contrary to the models that couple DNA packaging to head expansion, in T4 the expansion and packaging appear to be independent, since the already expanded DNA-free proheads can be packaged in vitro. We therefore propose that the unexpanded to expanded prohead transition has evolved to stabilize the capsid and to reorganize the prohead shell functionally from a core-interacting to a DNA-interacting inner surface.

The naturally occurring polycationic polyamines including putrescine, spermidine, and spermine play an important role in cell growth, differentiation, and gene expression. However, circulating polyamines are potential substrates for several oxidizing enzymes including copper-containing serum amine oxidase. These enzymes are capable of oxidizing serum polyamines to several toxic metabolites including aldehydes and H(2)O(2). In this study, we investigated the effects of polyamines as inducers of phase 2 enzymes and other genes that promote cell survival in a cell culture system in the presence of bovine serum. Spermidine and spermine (50 microM) increased NAD(P)H quinone oxidoreductase (NQO1) activity up to 3-fold in murine keratinocyte PE cells. Transcript levels for glutathione S-transferase (GST) A1, GST M1, NQO1, gamma-glutamylcysteine ligase regulatory subunit, and UDP-glucuronyltransferase 1A6 were significantly increased by spermidine and this effect was mediated through the antioxidant response element (ARE). The ARE from the mouse GST A1 promoter was activated about 9-fold by spermine and 5-fold by spermidine treatment, but could be inhibited by the amine oxidase inhibitor, aminoguanidine, suggesting that acrolein or hydrogen peroxide generated from polyamines by serum amine oxidase may be mediators for phase 2 enzyme induction. Elevations of ARE-luciferase expression and NQO1 enzyme activity by spermidine were not affected by catalase, while both were completely repressed by aldehyde dehydrogenase treatment. Direct addition of acrolein to PE cells induced multiple phase 2 genes and elevated nuclear levels of Nrf2, a transcription factor that binds to the ARE. Expression of mutant Nrf2 repressed the activation of the ARE-luciferase reporter by polyamines and acrolein. These results indicate that spermidine and spermine increase the expression of phase 2 genes in cells grown in culture through activation of the Nrf2-ARE pathway by generating the sulfhydryl reactive aldehyde, acrolein.

In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide.

A detailed study of the mechanism of nascent chain elongation and of steady state kinetics of purified mouse DNA polymerase alpha has been conducted. Polymerization was examined using a model replication system of poly(dT) as template, oligo(rA) as primer, and dATP as nucleotide substrate, and the probability of chain termination was determined by measurement of the precise chainlength of the products. Reactions were conducted under conditions where products were not utilized as primer. Product chainlength analysis indicated that alpha-polymerase acted in a processive fashion, elongating the primer by the stepwise addition of up to 20 dAMP residues before dissociating. The probability of termination after each dAMP addition depended upon the chainlength of the product and upon the presence of several agents; spermine, spermidine, putrescine, nalidixic acid, or PPi caused a marked increase in termination after the first dAMP addition, and conversely, mouse helix destabilizing protein-1 caused the enzyme to continue extending the same product chain until 18 to approximately 35 dAMP residues had been added. From these and other data, it is concluded that the kinetic mechanisms of termination after the first dAMP addition and after subsequent dAMP additions are different. With this information on how alpha-polymerase elongates a nascent primer(dA)n molecule, a kinetic model and appropriate steady state rate equations were obtained for analysis of substrate initial velocity data and termination probabilities. The substrate kinetic patterns and PPi product inhibition results were consistent with the ordered Ter Ter mechanism Bi Uni Uni Bi Ping Pong proposed in the model, and the model also permits a rational explanation for the differences in termination probability and for the fact that substrate initial velocity plots were linear even though multiple residues of dATP combined with the enzyme during each catalytic cycle. In addition, the results suggest that a rate-limiting step in the steady state occurs at the transition between initiation and elongation, and that higher levels of template.primer increase the rate of this step. This secondary effect of template.primer is discussed in relation to other polymer-forming enzymes, and various kinetic mechanisms which require the presence of two template.primer-binding sites, effector and catalytic, are discussed for their fit to the experimental data.

Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy, and heart failure. The polyamines putrescine, spermidine, and spermine are polycations absolutely required for cell growth and division. However, increasing evidence indicates that polyamines, cell growth, and cell death can be tightly connected. In this paper, we have studied the involvement of polyamines in apoptosis of H9c2 cardiomyoblasts in a model of simulated ischemia. H9c2 cells were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation, that induces apoptosis. The activity of ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis that synthesizes putrescine, is rapidly and transiently induced in ischemic cells, reaching a maximum after 3 h, and leading to increased polyamine levels. Pharmacological inhibition of ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) depletes H9c2 cardiomyoblasts of polyamines and protects the cells against ischemia-induced apoptosis. DFMO inhibits several of the molecular events of apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, caspase activation, downregulation of Bcl-xL, and DNA fragmentation. The protective effect of DFMO is lost when exogenous putrescine is provided to the cells, indicating a specific role of polyamine synthesis in the development of apoptosis in this model of simulated ischemia. In cardiomyocytes obtained from transgenic mice overexpressing ornithine decarboxylase in the heart, caspase activation is dramatically increased following induction of apoptosis, with respect to cardiomyocytes from control mice, confirming a proapoptotic effect of polyamines. It is presented for the first time evidence of the involvement of polyamines in apoptosis of ischemic cardiac cells and the beneficial effect of DFMO treatment. In conclusion, this finding may suggest novel pharmacological approaches for the protection of cardiomyocytes injury caused by ischemia.

Socransky, S. S. (Forsyth Dental Center, Boston, Mass.), W. J. Loesche, C. Hubersak, and J. B. Macdonald. Dependency of Treponema microdentium on other oral organisms for isobutyrate, polyamines, and a controlled oxidation-reduction potential. J. Bacteriol. 88:200-209. 1964.-Strains of Treponema microdentium can be cultivated on a variety of autoclaved commercially available media in the presence of other oral organisms. Organisms supporting growth in these circumstances include a facultative diphtheroid accompanied by either a strain of Fusobacterium or a motile gram-negative anaerobic rod. Culture filtrates and lysates of these "supporting organisms" failed to substitute for growing organisms. Measurement of the oxidation-reduction potential of the test system demonstrated that the spirochetes grew in a narrow range of Eh (optimum, -190 mv). The supporting organisms could be replaced by their filtrates when the Eh of the medium was poised in this range by a combination of reducing agents. Both filtrates contained a heat-labile factor required by the spirochete, which could be replaced by 5 mug/ml of cocarboxylase. Isobutyric acid, which could be detected in the fusiform filtrate, and putrescine which could be detected in the diphtheroid filtrate, replaced the spirochete's remaining filtrate requirement. Maximal growth occurred when any of the following were incorporated into the medium: 2 mug/ml of sodium isobutyrate; 250 mug/ml of putrescine dihydrochloride; 200 mug/ml of spermidine phosphosphate, or 150 mug/ml of spermine tetrahydrochloride.

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Delta adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.