The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

In trypanosomes, the parasite-specific thiol trypanothione [T(SH)2] fulfills various functions, the best established being detoxification of H2O2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH)2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH)2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH)2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H2O2 and tert.-butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-conjugated thiols (Gsp and T(SH)2) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites.

L-arginine is a semi-essential amino acid with a number of bioactive metabolites. Accumulating evidence suggests the implication of altered arginine metabolism in the pathogenesis of Alzheimer's disease (AD). The present study systematically compared the metabolic profile of L-arginine in the superior frontal gyrus, hippocampus, and cerebellum from AD (mean age 80 years) and normal (mean age 80 or 60 years) cases. The activity and protein expression of nitric oxide synthase and arginase were altered with AD and age in a region-specific manner. There were also AD- and age-related changes in the tissue concentrations of L-arginine and its downstream metabolites (L-citrulline, L-ornithine, agmatine, putrescine, spermidine, spermine, glutamate, γ-aminobutyric acid, and glutamine) in a metabolite- or region-specific manner. These findings demonstrate that arginine metabolism is dramatically altered in diverse regions of AD brains, thus meriting further investigation to understand its role in the pathogenesis and/or progression of the disease.

N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.

Activation of cell-surface receptors often evokes changes in Ca2+ fluxes leading to an increase in cytosolic Ca2+, a generally accepted mediator of many cell responses. The molecular mechanisms by which surface agonists elicit these changes in Ca2+ flux have remained elusive. An increase in the polyamines putrescine, spermidine and spermine, and their rate-regulating, synthetic enzyme ornithine decarboxylase (ODC), is one of the earliest events that occur during cell growth, replication and differentiation. However, the precise physiological roles of the polyamines remain enigmatic. Recently, we found that testosterone induces an early (less than 60s), Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport and amino acid transport in mouse kidney cortex involving the proximal tubules. This response is associated with increased Ca2+ fluxes and a mobilization of intracellular calcium, and is thought to represent a direct, receptor-mediated action of testosterone on the surface membrane. Polyamine synthesis was previously found to be essential for the long-term effects of testosterone on mouse kidney. We now report that testosterone evokes a rapid (less than 30 s), transient increase in ODC activity and a sustained increase in polyamines in kidney cortex. This polyamine synthesis is obligatory for stimulation of membrane transport functions and Ca2+ fluxes. These findings form the basis for a new theory of information flow in stimulus-response coupling in which the polyamines serve as messengers to generate a Ca2+ signal by increasing Ca2+ influx and mobilizing intracellular calcium via a cation-exchange reaction.

Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 microM 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by approximately 5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.

Stenotrophomonas rhizophila has great potential for applications in biotechnology and biological control due to its ability to both promote plant growth and protect roots against biotic and a-biotic stresses, yet little is known about the mode of interactions in the root-environment system. We studied mechanisms associated with osmotic stress using transcriptomic and microscopic approaches. In response to salt or root extracts, the transcriptome of S. rhizophila DSM14405(T) changed drastically. We found a notably similar response for several functional gene groups responsible for general stress protection, energy production, and cell motility. However, unique changes in the transcriptome were also observed: the negative regulation of flagella-coding genes together with the up-regulation of the genes responsible for biofilm formation and alginate biosynthesis were identified as a single mechanism of S. rhizophila DSM14405(T) against salt shock. However, production and excretion of glucosylglycerol (GG) were found as a remarkable mechanism for the stress protection of this Stenotrophomonas strain. For S. rhizophila treated with root exudates, the shift from the planktonic lifestyle to a sessile one was measured as expressed in the down-regulation of flagellar-driven motility. These findings fit well with the observed positive regulation of host colonization genes and microscopic images that show different colonization patterns of oilseed rape roots. Spermidine, described as a plant growth regulator, was also newly identified as a protector against stress. Overall, we identified mechanisms of Stenotrophomonas to protect roots against osmotic stress in the environment. In addition to both the changes in life style and energy metabolism, phytohormons, and osmoprotectants were also found to play a key role in stress protection.

The binding of the outer membrane-disorganizing peptide polymyxin B nonapeptide (PMBN) to gram-negative bacteria was studied by using tritium-labeled PMBN. Smooth Salmonella typhimurium had a binding capacity of ca. 6 nmol of PMBN per mg (dry weight) of bacteria, which corresponds to ca. 1 X 10(6) to 2 X 10(6) molecules of PMBN per single cell. The binding was of relatively high affinity (Kd, 1.3 microM). The isolated outer membrane of S. typhimurium bound ca. 100 nmol of PMBN per mg of outer membrane protein (Kd, 1.1 microM), whereas the cytoplasmic membrane bound 9 to 10 times less. Other bacteria which are susceptible to the action of PMBN (Escherichia coli strains, Pseudomonas aeruginosa, Haemophilus influenzae) also bound large amounts of PMBN. The S. typhimurium pmrA mutant, Neisseria gonorrhoeae, and Proteus mirabilis (all known as resistant to polymyxin and PMBN) bound 3.3, 4, and 12 times less than S. typhimurium, respectively. The binding of PMBN to S. typhimurium was effectively inhibited by low concentrations of polymyxin B, compound EM49 (octapeptin), polylysine, and protamine. Spermine, Ca2+, and Mg2+ also inhibited the PMBN binding although they were ca. 160, 700, and 2,400 times less active (based on molarity) than polymyxin B, respectively. No binding inhibition was found at the tested concentrations of streptomycin, tetralysine, spermidine, or cadaverine.

4-Aminobutyrate (GABA) accumulates in various plant parts, including bulky fruits such as apples, in response to abiotic stress. It is generally believed that the GABA is derived from glutamate, although a contribution from polyamines is possible. Putrescine, but not spermidine and spermine, generally accumulates in response to the genetic manipulation of polyamine biosynthetic enzymes and abiotic stress. However, the GABA levels in stressed plants are influenced by processes other than putrescine availability. It is hypothesized that the catabolism of putrescine to GABA is regulated by a combination of gene-dependent and -independent processes. The expression of several putative diamine oxidase genes is weak, but highly stress-inducible in certain tissues of Arabidopsis. In contrast, candidate genes that encode 4-aminobutyraldehyde dehydrogenase are highly constitutive, but not stress inducible. Changes in O(2) availability and cellular redox balance due to stress may directly influence the activities of diamine oxidase and 4-aminobutyraldehyde dehydrogenase, thereby restricting GABA formation. Apple fruit is known to accumulate GABA under controlled atmosphere storage and therefore could serve as a model system for investigating the relative contribution of putrescine and glutamate to GABA production.

Familial adenomatous polyposis (FAP), due to germ-line mutation of the adenomatous polyposis coli (APC) gene, is characterized by development of colorectal adenomas and ultimately colorectal cancer. The usefulness of ornithine decarboxylase (ODC) activity and polyamine levels in normal-appearing colorectal mucosa to stratify risk for colorectal neoplasia by discriminating presymptomatic individuals with germ-line APC mutation (genotype-positive) from genotype-negative family controls was evaluated in 36 at-risk subjects undergoing endoscopic and genetic screening for FAP. ODC activity and levels of putrescine, spermidine, and spermine were significantly higher in presymptomatic genotype-positive patients compared to genotype-negative persons (P = 0.029, <0.001, 0.002, and <0.001, respectively). Moreover, a putrescine level with a cutoff point of 1.5 nmol/mg protein was the most accurate single discriminator of risk status. ODC activity and polyamine levels are significantly elevated in gene carriers of FAP before the development of polyposis, suggesting a role for these compounds in tumorigenesis of FAP. These assays may be useful in evaluating at-risk members of FAP families in which mutation of the APC gene cannot be found.

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, Y. P., and Chen, K. Y. (1997) Biol. Signals 6, 166-174). Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) m. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in>> 400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5'-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.

Progression of Parkinson's disease (PD) is highly variable, indicating that differences between slow and rapid progression forms could provide valuable information for improved early detection and management. Unfortunately, this represents a complex problem due to the heterogeneous nature of humans in regards to demographic characteristics, genetics, diet, environmental exposures and health behaviors. In this pilot study, we employed high resolution mass spectrometry-based metabolic profiling to investigate the metabolic signatures of slow versus rapidly progressing PD present in human serum. Archival serum samples from PD patients obtained within 3 years of disease onset were analyzed via dual chromatography-high resolution mass spectrometry, with data extraction by xMSanalyzer and used to predict rapid or slow motor progression of these patients during follow-up. Statistical analyses, such as false discovery rate analysis and partial least squares discriminant analysis, yielded a list of statistically significant metabolic features and further investigation revealed potential biomarkers. In particular, N8-acetyl spermidine was found to be significantly elevated in the rapid progressors compared to both control subjects and slow progressors. Our exploratory data indicate that a fast motor progression disease phenotype can be distinguished early in disease using high resolution mass spectrometry-based metabolic profiling and that altered polyamine metabolism may be a predictive marker of rapidly progressing PD.

BACKGROUND

Spermine synthase (SMS) is a key enzyme controlling the concentration of spermidine and spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable.

RESULTS

To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement.

CONCLUSIONS

The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs can be expected to occur even at sites at which missense mutations were found to cause diseases.

Multidrug transporters pump structurally dissimilar toxic molecules out of cells. It is not known, however, if detoxification is the primary physiological function of these transporters. The chromosomal organization of the gene encoding the Bacillus subtilis multidrug transporter Blt suggests a specific function for this protein; it forms a single operon with another gene, bltD, whose protein product is identified here as a spermine/spermidine acetyltransferase, an enzyme catalyzing a key step in spermidine degradation. Overexpression of the Blt transporter in B. subtilis leads not only to the multidrug-resistance phenotype but also to the efflux of large amounts of spermidine into the medium; this efflux is supressed by an inhibitor of Blt, reserpine. Taken together, these results strongly suggest that the natural function of the Blt transporter is the efflux of spermidine, whereas multiple drugs may be recognized by Blt merely opportunistically.

Single-strand gaps in DNA molecules were found to be a substrate for T4 DNA ligase. Sealing of the gaps was optimal at the same conditions as ligation of blunt-ended DNA molecules. Spermidine at a concentration of 2 mM stimulated the ligation of gaps, as well as the joining of DNA molecules with cohesive and blunt ends. In addition, spermidine reduced the optimal ATP concentration. The ligation of single-stranded gaps was a slow process, reaching a plateau after several hours at 25 degrees C. Approximately 10% of circular duplex plasmid pBR322 DNA molecules with a gap of 1-5 nucleotides could be converted to a covalently closed form. When such molecules were used for transformation of E. coli cells deletion mutants were obtained at a high frequency. The size and position of the gaps and the deletions were equivalent, confirming that T4 DNA ligase was sealing the gaps.

(14)C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3-6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [(14)C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [(14)C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a "steam girdle" translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.

Polyamine oxidase (PAO) catalyzes the oxidative catabolism of spermidine and spermine, generating hydrogen peroxide. In wild-type tobacco (Nicotiana tabacum 'Xanthi') plants, infection by the compatible pathogen Pseudomonas syringae pv tabaci resulted in increased PAO gene and corresponding PAO enzyme activities; polyamine homeostasis was maintained by induction of the arginine decarboxylase pathway and spermine was excreted into the apoplast, where it was oxidized by the enhanced apoplastic PAO, resulting in higher hydrogen peroxide accumulation. Moreover, plants overexpressing PAO showed preinduced disease tolerance against the biotrophic bacterium P. syringae pv tabaci and the hemibiotrophic oomycete Phytophthora parasitica var nicotianae but not against the Cucumber mosaic virus. Furthermore, in transgenic PAO-overexpressing plants, systemic acquired resistance marker genes as well as a pronounced increase in the cell wall-based defense were found before inoculation. These results reveal that PAO is a nodal point in a specific apoplast-localized plant-pathogen interaction, which also signals parallel defense responses, thus preventing pathogen colonization. This strategy presents a novel approach for producing transgenic plants resistant to a broad spectrum of plant pathogens.

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.

As soon as the natural polyamines (PAs), putrescine (Put), spermidine (Spd) and spermine (Spm), were recognized as ubiquitous constituents of eukaryotic cells, their involvement in growth-related processes attracted particular interest. The high activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) in rapidly growing tissues and cells, particularly in tumour cells, suggested PA biosynthesis as a target for antineoplastic therapy. In the course of the years selective inhibitors have been developed for literally all enzymes of PA metabolism. Some became important as tools in the elucidation of the PA metabolic system, but only few of them were efficient as inhibitors of tumour growth. A major reason for the inefficacy of selective enzyme inhibitors as anticancer drugs is the sophistication of the system, which regulates intracellular PA pools. Selective blockade of a single enzyme induces changes of metabolism and transport, which compensate for the deficit. The selective impairment of tumour growth is in addition hampered by the ubiquitous occurrence of the PAs, their importance in normal functions of nearly all mammalian cells, and the ability or the mammalian organism to utilize exogenous (gastrointestinal) PAs. Among the inhibitors of PA-related enzymes, the ODC inactivator (R, S)-2-(difluoromethyl)ornithine (DFMO) became most famous. Although it was disappointing in most therapeutic attempts to use it as single drug, it has--based on its low toxicity--considerable potential in cancer chemoprevention, and it turned out to be a highly efficient anti-trypanosome agent. Very likely DFMO is suitable to improve the efficacy of some of the current cytotoxic drugs, and it may allow one to create new therapies in combination with other PA-directed drugs. Some of the less selective enzyme inhibitors, particularly those, which inhibit two or more enzymes of PA metabolism, appear to have had a chance to become practically useful, but they have not been developed energetically. Disregarding DFMO, the AdoMetDC inhibitor SAM486A is the only compound for which clinical trials were published. The future of this drug is unclear at present; presumably phase III clinical trials have been discontinued. One of the lessons that had to be learned from the work on selective enzyme inhibitors was that PA metabolism is a much more difficult target, than has been expected on the basis of the simplicity of the PA structures, and the simple reactions involved in their biosynthesis. In order to inhibit tumour growth several reactions or regulatory functions of PA metabolism have to be impaired at the same time. Recent efforts devoted to the development new types of anticancer drugs, which are based on the perturbation of PA metabolism by structural analogues of the natural PAs, take this message into account. These approaches are the topic of the 2nd part of this overview.

The enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) regulates the catabolism and export of intracellular polyamines. We have previously shown that activation of polyamine catabolism by conditional overexpression of SSAT has antiproliferative consequences in LNCaP prostate carcinoma cells. Growth inhibition was causally linked to high metabolic flux arising from a compensatory increase in polyamine biosynthesis. Here we examined the in vivo consequences of SSAT overexpression in a mouse model genetically predisposed to develop prostate cancer. TRAMP (transgenic adenocarcinoma of mouse prostate) female C57BL/6 mice carrying the SV40 early genes (T/t antigens) under an androgen-driven probasin promoter were cross-bred with male C57BL/6 transgenic mice that systemically overexpress SSAT. At 30 weeks of age, the average genitourinary tract weights of TRAMP mice were approximately 4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were approximately 12 times greater indicating sustained suppression of tumor outgrowth. Tumor progression was also affected as indicated by a reduction in the prostate histopathological scores. By immunohistochemistry, SV40 large T antigen expression in the prostate epithelium was the same in TRAMP and TRAMP/SSAT mice. Consistent with the 18-fold increase in SSAT activity in the TRAMP/SSAT bigenic mice, prostatic N(1)-acetylspermidine and putrescine pools were remarkably increased relative to TRAMP mice, while spermidine and spermine pools were minimally decreased due to a compensatory 5-7-fold increase in biosynthetic enzymes activities. The latter led to heightened metabolic flux through the polyamine pathway and an associated approximately 70% reduction in the SSAT cofactor acetyl-CoA and a approximately 40% reduction in the polyamine aminopropyl donor S-adenosylmethionine in TRAMP/SSAT compared with TRAMP prostatic tissue. In addition to elucidating the antiproliferative and metabolic consequences of SSAT overexpression in a prostate cancer model, these findings provide genetic support for the discovery and development of specific small molecule inducers of SSAT as a novel therapeutic strategy targeting prostate cancer.

With the advent of high-throughput DNA sequencing, it is now straightforward and inexpensive to generate high-density small nucleotide polymorphism (SNP) maps. Here we combined high-throughput sequencing with bulk segregant analysis to expedite mutation mapping. The general map location of a mutation can be identified by a single backcross to a strain enriched in SNPs compared to a standard wild-type strain. Bulk segregant analysis simultaneously increases the likelihood of determining the precise nature of the mutation. We present here a high-density SNP map between Neurospora crassa Mauriceville-1-c (FGSC2225) and OR74A (FGSC2489), the strains most typically used by Neurospora researchers to carry out mapping crosses. We further have demonstrated the utility of the Mauriceville sequence and our approach by mapping the mutation responsible for the only existing temperature-sensitive (ts) cell cycle mutation in Neurospora, nuclear division cycle-1 (ndc-1). The single T-to-C point mutation maps to the gene encoding ornithine decarboxylase (ODC), spe-1 (NCU01271), and changes a Phe to a Ser residue within a highly conserved motif next to the catalytic site of the enzyme. By growth on spermidine and complementation with a wild-type spe-1 gene, we showed that the defect in spe-1 is responsible for the ts ndc-1 mutation. Based on our results, we propose changing ndc-1 to spe-1(ndc), which reflects that this mutation results in an ODC with a specific nuclear division defect.

The spermidine in rat liver increases after partial hepatectomy, and the rate of polyamine accumulation closely approximates the increased rate of synthesis of RNA in regenerating liver. The uptake by the liver of intravenously injected putrescine and the biosynthesis of spermidine are accelerated within 2 hours after the operation. The uptake of spermidine also increases during early regeneration.