In recent years, our understanding of the importance of membrane transporters (MTs) in the disposition of and response to drugs has increased significantly. MTs are proteins that regulate the transport of endogenous molecules and xenobiotics across the cell membrane. In mammals, two super-families have been identified: ATP-binding cassette (ABC) and solute carrier (SLC) transporters. There is evidence that MTs might mediate polyamines (PA) transport. PA are ubiquitous polycations which are found in all living cells. In mammalian cells, three major PA are synthesised: putrescine, spermidine and spermine; whilst the decarboxylated arginine (agmatine) is not produced by mammals but is synthesised by plants and bacteria. In addition, research in the PA field suggests that PA are transported into cells via a specific transporter, the polyamine transport system(s) (PTS). Although the PTS has not been fully defined, there is evidence that some of the known MTs might be involved in PA transport. In this mini review, eight SLC transporters will be reviewed and their potential to mediate PA transport in human cells discussed. These transporters are SLC22A1, SLC22A2, SLC22A3, SLC47A1, SLC7A1, SLC3A2, SLC12A8A, and SLC22A16. Preliminary data from our laboratory have revealed that SLC22A1 might be involved in the PA uptake; in addition to one member of ABC superfamily (MDR1 protein) might also mediate the efflux of polyamine like molecules.

The acute and subacute toxicity of five biogenic amines-tyramine, spermidine, spermine, putrescine and cadaverine-were examined in Wistar rats. Tyramine and cadaverine had a low acute oral toxicity of more than 2000 mg/kg body weight. Putrescine had an acute oral toxicity of 2000 mg/kg body weight and spermidine and spermine each of 600 mg/kg body weight. All amines investigated caused a dose-related decrease in blood pressure after intravenous administration, except for tyramine, where an increase was found. In 6-wk studies the biogenic amines were administered in the diet to groups of 10 male and 10 female rats. Tyramine and cadaverine were given at levels of 0, 200, 2000 or 10,000 ppm, spermine and putrescine at levels of 0, 200, 2000 or 5000 ppm and spermidine at levels of 0, 20, 200 or 500/1000 ppm in the first study and at levels of 0 or 10,000 ppm in a second study. Spermine was the most toxic. The high dose level showed a great number of changes, such as emaciation, aggressiveness, convulsions and paralysis of the hind legs. Growth, food intake and water intake were considerably decreased. Slight anaemia (males) and changes in plasma clinical chemistry occurred. The relative weights of the thyroid, adrenals, spleen and heart were increased and that of the liver decreased. Impaired kidney function, together with renal histopathological changes and changes in plasma electrolytes and urea, occurred with spermine. Histopathological examinations also revealed decreased glycogen content in the liver, reduction of spermatogenesis, severe depletion of splenic white pulp, acute involution of the thymus and moderate myocardial degeneration in the heart. Myocardial degeneration was also seen in one mid-dose male. Adverse effects were also observed in the top dose groups of all other amines. Decreased body weights associated with diminished food intake were generally seen. Slight increases in packed cell volume, haemoglobin concentration and thrombocytes occurred with cadaverine. With spermidine, decreased plasma creatinine, calcium and inorganic phosphate were observed and decreased potassium levels with cadaverine. The no-observed-adverse-effect level was 2000 ppm (180 mg/kg body weight/day) for tyramine, cadaverine and putrescine, 1000 ppm (83 mg/kg body weight/day) for spermidine and 200 ppm (19 mg/kg body weight/day) for spermine.

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense.

The main kidney transporter of many commonly prescribed drugs (e.g. penicillins, diuretics, antivirals, methotrexate, and non-steroidal anti-inflammatory drugs) is organic anion transporter-1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478). Targeted metabolomics in knockouts have shown that OAT1 mediates the secretion or reabsorption of many important metabolites, including intermediates in carbohydrate, fatty acid, and amino acid metabolism. This observation raises the possibility that OAT1 helps regulate broader metabolic activities. We therefore examined the potential roles of OAT1 in metabolic pathways using Recon 1, a functionally tested genome-scale reconstruction of human metabolism. A computational approach was used to analyze in vivo metabolomic as well as transcriptomic data from wild-type and OAT1 knock-out animals, resulting in the implication of several metabolic pathways, including the citric acid cycle, polyamine, and fatty acid metabolism. Validation by in vitro and ex vivo analysis using Xenopus oocyte, cell culture, and kidney tissue assays demonstrated interactions between OAT1 and key intermediates in these metabolic pathways, including previously unknown substrates, such as polyamines (e.g. spermine and spermidine). A genome-scale metabolic network reconstruction generated some experimentally supported predictions for metabolic pathways linked to OAT1-related transport. The data support the possibility that the SLC22 and other families of transporters, known to be expressed in many tissues and primarily known for drug and toxin clearance, are integral to a number of endogenous pathways and may be involved in a larger remote sensing and signaling system (Ahn, S. Y., and Nigam, S. K. (2009) Mol. Pharmacol. 76, 481-490, and Wu, W., Dnyanmote, A. V., and Nigam, S. K. (2011) Mol. Pharmacol. 79, 795-805). Drugs may alter metabolism by competing for OAT1 binding of metabolites.

Oral cancers are the sixth most frequent cancer with a high mortality rate. Oral squamous cell carcinoma accounts for more than 90% of all oral cancers. Standard methods used to detect oral cancers remain comprehensive clinical examination, expensive biochemical investigations, and invasive biopsy. The identification of biomarkers from biological fluids (blood, urine, saliva) has the potential of early diagnosis. The use of saliva for early cancer detection in the search for new clinical markers is a promising approach because of its noninvasive sampling and easy collection methods. Human whole-mouth saliva contains proteins, peptides, electrolytes, organic, and inorganic salts secreted by salivary glands and complimentary contributions from gingival crevicular fluids and mucosal transudates. This diagnostic modality in the field of molecular biology has led to the discovery and potential of salivary biomarkers for the detection of oral cancers. Biomarkers are the molecular signatures and indicators of normal biological, pathological process, and pharmacological response to treatment hence may provide useful information for detection, diagnosis, and prognosis of the disease. Saliva's direct contact with oral cancer lesions makes it more specific and potentially sensitive screening tool, whereas more than 100 salivary biomarkers (DNA, RNA, mRNA, protein markers) have already been identified, including cytokines (IL-8, IL-1b, TNF-α), defensin-1, P53, Cyfra 21-1, tissue polypeptide-specific antigen, dual specificity phosphatase, spermidine/spermineN1-acetyltransferase , profilin, cofilin-1, transferrin, and many more. However, further research is still required for the reliability and validation of salivary biomarkers for clinical applications. This chapter provides the latest up-to-date list of known and emerging potential salivary biomarkers for early diagnosis of oral premalignant and cancerous lesions and monitoring of disease activity.

Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.

Twelve young adult (1.7 +/- 0.1 yr) male cats were used in a replicated 3 x 3 Latin square design to determine the effects of fiber type on nutrient digestibility, fermentative end products, and fecal microbial populations. Three diets containing 4% cellulose, fructooligosaccharides (FOS), or pectin were evaluated. Feces were scored based on the 5-point system: 1 being hard, dry pellets, and 5 being watery liquid that can be poured. No differences were observed (P>> 0.100) in intake of DM, OM, CP, or acid-hydrolyzed fat; DM or OM digestibility; or fecal pH, DM%, output on an as-is or DM basis, or concentrations of histamine or phenylalanine. Crude protein and fat digestibility decreased (P = 0.079 and 0.001, respectively) in response to supplementation with pectin compared with cellulose. Both FOS and pectin supplementation resulted in increased fecal scores (P < 0.001) and concentrations of ammonia (P = 0.003) and 4-methyl phenol (P = 0.003). Fecal indole concentrations increased (P = 0.049) when cats were supplemented with FOS. Fecal acetate (P = 0.030), propionate (P = 0.035), and total short-chain fatty acid (P = 0.016) concentrations increased in pectin-supplemented cats. Fecal butyrate (P = 0.010), isobutyrate (P = 0.011), isovalerate (P = 0.012), valerate (P = 0.026), and total branched-chain fatty acids + valerate (P = 0.008) concentrations increased with supplementation of FOS and pectin. Fecal cadaverine (P < 0.001) and tryptamine (P < 0.001) concentrations increased with supplementation of FOS and pectin. Fecal tyramine concentrations decreased (P = 0.039) in FOS-supplemented cats, whereas spermidine concentrations increased (P < 0.001) in pectin-supplemented cats. Whereas fecal concentrations of putrescine (P < 0.001) and total biogenic amines (P < 0.001) increased with FOS and pectin, the concentrations of these compounds were increased (P < 0.001) in cats supplemented with pectin. Fecal Bifidobacterium spp. concentrations increased (P = 0.006) and Escherichia coli concentrations decreased (P < 0.001) in FOS-supplemented cats. Fecal concentrations of Clostridium perfringens (P < 0.001), E. coli (P < 0.001), and Lactobacillus spp. (P = 0.030) also increased in pectin-supplemented cats. In addition to increasing populations of protein-fermenting microbiota, pectin increased production of fermentative end products associated with carbohydrate compared with protein fermentation. Pectin and FOS may be useful fiber sources in promoting intestinal health of the cat.

A transport system for polyamines was studied with both intact cells and membrane vesicles of an Escherichia coli polyamine-deficient mutant. Polyamine uptake by intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artificially by the addition of valinomycin to K+-loaded vesicles. These results show that the uptake was dependent on proton motive force. Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. However, they were excreted by membrane vesicles, although the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, thus giving rise to a negligible free intracellular concentration of polyamines. Polyamine uptake, especially putrescine uptake, was inhibited strongly by monovalent cations. The Mg2+ ion inhibited spermidine and spermine uptake but not putrescine uptake.

Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines and have important roles in maintaining normal cellular function, regulating neurotransmitter receptors and modulating learning and memory. Recent evidence suggests a role of putrescine in hippocampal neurogenesis, that is significantly impaired during aging. The present study measured the polyamine levels in memory-related brain structures in 24- (aged), 12- (middle-aged) and 4- (young) month-old rats using liquid chromatography/mass spectrometry and high performance liquid chromatography. In the hippocampus, the putrescine levels were significantly decreased in the CA1 and dentate gyrus, and increased in the CA2/3 with age. Significant age-related increases in the spermidine levels were found in the CA1 and CA2/3. There was no difference between groups in spermine in any sub-regions examined. In the parahippocampal region, increased putrescine level with age was observed in the entorhinal cortex, and age did not alter the spermidine levels. The spermine level was significantly decreased in the perirhinal cortex and increased in the postrhinal cortex with age. In the prefrontal cortex, there was age-related decrease in putrescine, and the spermidine and spermine levels were significantly increased with age. This study, for the first time, demonstrates age-related region-specific changes in polyamines in memory-associated structures, suggesting that polyamine system dysfunction may potentially contribute to aged-related impairments in hippocampal neurogenesis and learning and memory.

The polyamines, spermidine and spermine, were first discovered in 1678 by Antonie van Leeuwenhoek. In the early part of the 20th century their structure was determined and their pathway of biosynthesis established. The polyamines are essential elements of cells from all species. They are required for optimum cell growth, and cells where polyamine production has been prevented by mutation, or blocked by inhibitors, require exogenous provision of at least one polyamine for continued survival. Despite this critical function, the polyamines have not attracted as much attention as they deserve in the wider field of biochemistry and cell biology. They are rarely mentioned in standard textbooks, despite over 75000 research papers having been written on the subject since 1900, and more than half (54%) were published after 1990. There have been a number of books dedicated to the polyamines published and "The Guide to the Polyamines" by Seymour Cohen deserves mention as a work of outstanding scholarship describing "everything you ever wanted to know about the polyamines" in exquisite detail. The current volume of Essays in Biochemistry has a much humbler aim: to introduce the polyamines to interested researchers and students, and to describe how they are associated with, and might be utilized as a target for intervention in major diseases such as cancer.

Histatin 5 (Hst 5) is a salivary human antimicrobial peptide that is toxic to the opportunistic yeast Candida albicans. Fungicidal activity of Hst 5 requires intracellular translocation and accumulation to a threshold concentration for it to disrupt cellular processes. Previously, we observed that total cytosolic levels of Hst 5 were gradually reduced from intact cells, suggesting that C. albicans possesses a transport mechanism for efflux of Hst 5. Since we identified C. albicans polyamine transporters responsible for Hst 5 uptake, we hypothesized that one or more polyamine efflux transporters may be involved in the efflux of Hst 5. C. albicans FLU1 and TPO2 were found to be the closest homologs of Saccharomyces cerevisiae TPO1, which encodes a major spermidine efflux transporter, indicating that the products of these two genes may be involved in efflux of Hst 5. We found that flu1Δ/Δ cells, but not tpo2Δ/Δ cells, had significant reductions in their rates of Hst 5 efflux and had significantly higher cytoplasmic Hst 5 and Hst 5 susceptibilities than did the wild type. We also found that flu1Δ/Δ cells had reduced biofilm formation compared to wild-type cells in the presence of Hst 5. Transcriptional levels of FLU1 were not altered over the course of treatment with Hst 5; therefore, Hst 5 is not likely to induce FLU1 gene overexpression as a potential mechanism of resistance. Thus, Flu1, but not Tpo2, mediates efflux of Hst 5 and is responsible for reduction of its toxicity in C. albicans.

The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle cerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 +/- 0.3 to 84.5 +/- 0.9% in cortex (p < 0.05) and from 79.9 +/- 1.7 to 78.4 +/- 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex (p < 0.05) and 17% in the subcortex (p < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site after ischemia-reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase in putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.

Previous work has described the novel ability to modulate in vitro the activity of restriction endonuclease NaeI from Nocardia aerocoligenes by using cleavable DNA and spermidine [Conrad & Topal (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. In this paper we report the results of a study of 49 type II restriction enzymes from a variety of bacterial species. On the basis of the rates of cleavage observed, we found that in addition to expected cleavable sites a number of enzymes had slow and resistant cognate recognition sites. Resistant sites were identified for BspMI, NaeI, and NarI; slow sites were identified for HpaII, NaeI, and SacII. Cleavage of these sites was found to be significantly enhanced by the addition of cleavable DNA or spermidine. We demonstrate that for BspMI, as for NaeI, activator DNAs increased Vmax without altering Km, whereas for HpaII, NarI, and SacII activator DNAs decreased Km without changing Vmax. Comparison among the Kms for NaeI cleavage of several different substrates demonstrated that distant DNA sequences can affect DNA recognition by the activated enzyme. Our observations extend DNA activation of the Nocardia NaeI endonuclease to restriction endonucleases from Nocardia argentinensis (NarI), Bacillus species M (BspMI), Haemophilus parainfluenza (HpaII), and Streptomyces achromogenes (SacII). In addition, activation has now been found to affect slow as well as resistant recognition sites.

The exchangeable N1 imino protons of two pseudouridine (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC psi psi ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi N1 imino protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H2O (1). These protons will exchange readily at increased pH values or upon thermal denaturation of the duplex. The longitudinal (T1) relaxation times of the psi N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi N1 imino protons become readily exchangeable at a temperature some 20 degrees C below the melting temperature of the duplex.

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized "RzB" hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na(+) alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg(2+) and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min(-1) in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg(2+), unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg(2+) ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg(2+), low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg(2+) alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg(2+) alone. These observations suggest that Mg(2+) and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role.

Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of L-[3H]-glutamic acid (L-[3H]Glu) and DL-(E)-2-[3H]amino-4-propyl-5-phosphono-3- pentenoic acid ([3H]CGP-39653) in rat brain synaptic membranes, with less potent displacement of binding of DL-alpha-amino-3-hydroxy-5-[3H]-methylisoxazole-4-propionic and [3H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with L-Glu by < 1%. Both GSH and GSSG potentiated (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([3H]MK-801) binding in a manner similar to that found with L-Glu. Pretreatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration-response curve for L-Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [3H]MK-801 binding to a some-what smaller extent than that found in the presence of L-Glu at a maximally effective concentration. The potentiation of [3H]MK-801 binding by GSH was invariably attenuated by addition of CGP-39653, D-2-amino-5-phosphonovaleric acid (D-AP5), and 5,7-dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP-39653, D-AP5, DCKA, and 6,7-dichloroquinoxaline-2,3-dione to inhibit [3H]MK-801 binding when determined in the presence of both L-Glu and Gly.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutants of Escherichia coli and Saccharomyces cerevisiae that lack O6-alkylguanine-DNA alkyltransferase activities have increased spontaneous mutation rates, indicating the presence of a cellular metabolite that can alkylate DNA. Bacterially catalysed nitrosation has been implicated previously in producing the endogenous alkylating agent(s). Here, nitrosated polyamines and azaserine, a model compound for nitrosated peptides, are shown to be mutagenic to E. coli ada ogt mutants deficient in O6-alkylguanine-DNA alkyltransferase activity. The mutagenicity of azaserine may be explained by its ability to methylate DNA, whereas nitrosated spermidine causes DNA damage that is susceptible to both nucleotide excision repair and O6-alkylguanine-DNA alkyltransferase activity, which indicates the generation of more bulky DNA adducts. Nitrosated peptides and polyamines are therefore potential endogenous mutagens that are harmful particularly in O6-alkylguanine-DNA alkyltransferase deficient cells.

We have shown that toxicity of paraquat for Escherichia coli is increased over 10-fold in strains defective in the biosynthesis of spermidine compared to isogenic strains containing spermidine. The increased sensitivity of these spermidine-deficient mutants to paraquat is eliminated by growth in medium containing spermidine or by endogenous supplementation of spermidine by the use of a speE+D+ plasmid. No paraquat toxicity is seen in the absence of oxygen, even in amine-deficient strains, indicating that superoxide is the agent responsible for the increased toxicity. However, the specific mechanisms responsible for the increased paraquat toxicity in the spermidine-deficient mutants remain to be determined. The marked sensitivity to paraquat of E. coli deficient in spermidine is of particular interest, since such mutants have no other phenotypic properties that can be easily assayed. This increased sensitivity has been used as the basis of a convenient method for scoring for mutants in polyamine biosynthesis and for the detection of plasmids containing the biosynthetic genes.

Saccharomyces boulardii (S. boulardii) is a non-pathogenic biotherapeutic agent, widely prescribed in a lyophilized form in many countries over the world. S. boulardii acts as a shuttle liberating effective enzymes, proteins and trophic factors during its intestinal transit that improve host immune defenses, digestion, and absorption of nutrients. In addition, S. boulardii secretes during its intestinal transit polyamines, mainly spermine and spermidine that regulate gene expression and protein synthesis. In this review, we will focus on the interactions of the yeast with the host intestinal mucosa.

Bacillus subtilis synthesizes polyamines by decarboxylating arginine to agmatine, which is subsequently hydrolysed to putrescine. Spermidine is synthesized from putrescine and decarboxylated S-adenosylmethionine (dAdoMet). In Gram-negative bacteria and in eukaryotes, AdoMet is decarboxylated by an unusual 'pyruvoyl' AdoMet decarboxylase (SpeD), the catalytic pyruvoyl moiety of which is generated by serinolysis of an internal serine with self-cleavage of the protein at the upstream peptide bond. Neither the Gram-positive bacterial nor the archaeal counterpart of the Escherichia coli SpeD enzyme were known. We have identified the corresponding B. subtilis speD gene (formely ytcF). Heterologous expression of the cognate Methanococcus jannaschii protein, MJ0315, demonstrated that it displays the same activity as B. subtilis SpeD, indicating that spermidine biosynthesis in Gram-positive bacteria and in archaea follows a pathway very similar to that of Gram-negatives and eukarya. In B. subtilis, transcription of speD is modulated by spermidine and methionine. Its expression is high under usual growth conditions. In contrast, the SpeD protein self-cleaves slowly in vitro, a noticeable difference with its archaeal counterpart. Under certain growth conditions (minimal medium containing succinate and glutamate as a carbon source), speD is co-transcribed with gapB, the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme required for gluconeogenesis. This observation may couple polyamine metabolism to sulphur and carbon metabolism by a so far unknown mechanism.

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.

A sensitive method of polyamine estimation has been adapted to the study of the organic cations of small amounts of nucleic acid. A procedure utilizing phenol extraction, alcohol precipitation, and separation on Sephadex G100 has been devised for the isolation of tRNA at low ionic strength. The procedure is applicable to the isolation of tRNA from liter batches of bacterial culture. With these methods we have examined the polyamines of tRNA isolated from polyauxotrophic strains of E. coli incubated under various physiological conditions and have found the following: (1) The tRNA from relaxed bacteria (TAU rel) harvested during exponential growth is heterogeneous with respect to polyamine content. Some portions of the population contain about one mole of spermidine per mole of tRNA. Some putrescine and an unknown amine are also present in low concentration. (2) After exponential TAU rel is incubated with thymine and uracil in the absence of arginine, the tRNA population is far more homogeneous with respect to polyamine content. The various fractions contain two moles of spermidine per mole of tRNA and a small amount of putrescine. (3) After exponential TAU rel is incubated in the absence of both arginine and uracil, the polyamine pattern of the tRNA resembles that isolated from exponential cells. (4) The tRNA from stringent bacterias harvested during exponential growth is heterogeneous with respect to polyamine distribution and some fractions contain relatively high concentrations of the unknown amine.