The vacuoles of Neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-P. Vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. Thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. Polyphosphate was found to be the only macromolecular polyanion in vacuoles of normal or phosphate-depleted cells. Gel filtration experiments revealed that about half the polyphosphate of normal vacuoles is bound strongly by vacuolar spermidine, Mg2+, and Ca2+. The polyphosphate thus occupied was not available for basic amino acid binding. We have identified about 90% of the cations of isolated vacuoles; in addition to spermidine, Mg2+, and Ca2+, the cation pool consists mainly of arginine, ornithine, histidine, lysine, and Na+, with a small amount of K+. Isolated vacuoles appear to be almost wholly impermeable to all these ions, and in vivo, vacuoles appear to be highly selective in ion uptake by an active process. The interaction of basic amino acid with the available polyphosphate was found to reduce the chemical activity of the former. In keeping with this effect, cells with abnormally high basic amino acid-polyphosphate ratios displayed greatly swollen vacuoles, indicating considerable osmotic activity of the basic amino acids and their counterions under these conditions.

High potassium (50 mM) depolarization induces a rapid (less than 15 sec) increase in the levels of the polyamines putrescine, spermidine and spermine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+-dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of alpha-difluoromethylornithine. These results suggest that enhanced polyamine synthesis is required for potassium depolarized stimulation of synaptic function.

A mutant of Escherichia coli with reduced levels of biosynthetic arginine decarboxylase was isolated which required putrescine or spermidine for optimal growth. The stimulation of growth by putrescine was 1.5- to 3-fold depending upon the culture medium. Specificity studies supported the concept that the requirement was for spermidine or closely related polyamines, or for diamines which could be converted enzymatically to these compounds. The behavior of the macromolecular composition of the polyamine-starved cells appeared abnormal. The ribonucleic acid to protein and deoxyribonucleic acid to cell ratios in the starved cells were both higher than expected on the basis of their growth rate. The stable ribonucleic acid in the polyamine-limited cells appeared to be normal as judged from size distribution and degree of methylation. The relationship of these results to mechanisms for regulation of nucleic acid and protein synthesis in E. coli is discussed.

Soluble fibronectin of human plasma was specifically dissociated at neutral pH from gelatin-agarose by several cationic amino compounds, notably the polyamines spermine, spermidine and putrescine, the basic amino acid arginine, and amino sugars. The neutral and acidic amino acids and the N-acetylated derivatives of amino sugars tested were ineffective. Gel-filtration experiments demonstrated that [14C]spermidine bound to fibronectin but not to gelatin.

The presence of certain rpsL (strA) mutations in a strain of Escherichia coli that cannot synthesize putrescine or spermidine because of deletions in ornithine decarboxylase, arginine decarboxylase, and agmatine ureohydrolase, converts a partial requirement for polyamines for growth into an absolute requirement.

Recent research progress on polyamines in extreme thermophiles is reviewed. Extreme thermophiles produce two types of unique polyamines; one is longer polyamines such as caldopentamine and caldohexamine, and the other is branched polyamines such as tetrakis(3-aminopropyl)ammonium. The protein synthesis catalyzed by a cell-free extract of Thermus thermophilus, an extreme thermophile, required the presence of a polyamine and the highest activity was found in the presence of tetrakis(3-aminopropyl)ammonium. In vitro experiments, longer polyamines efficiently stabilized double stranded nucleic acids and a branched polyamine, tetrakis(3-aminropyl)ammonium, stabilized stem-and-loop structures. In T. thermophilus, polyamines are synthesized from arginine by a new metabolic pathway; arginine is converted to agmatine and then agmatine is aminopropylated to N(1)-aminopropylagmatine which is converted to spermidine by an enzyme coded by a gene homologous to speB (a gene for agmatinase). In this new pathway spermidine is not synthesized from putrescine. Reverse genetic studies indicated that the unique polyamines are synthesized from spermidine.

We recently found that the beta-adrenergic agonist 1-isoproterenol evokes a rapid (less than 5 min) Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport, and amino acid transport in mouse renal cortex involving proximal tubule cells. This response is associated with increased Ca2+ fluxes and a mobilization of mitochondrial calcium, suggesting that stimulus-response (stimulus-"transport") coupling is mediated by cytosolic Ca2+. We show here that 1 microM isoproterenol evokes a rapid (less than 60 sec) transient increase in the activity of ornithine decarboxylase followed by an early (less than 2 min) sustained increase in putrescine, spermidine, and spermine concentrations in mouse kidney cortex slices in vitro. Small doses of isoproterenol (down to 24 nmol/kg) elicited a rapid (less than 2 min) increase in polyamines in vivo. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine (5 mM) suppressed the testosterone-induced increase in polyamine levels and rates of endocytosis, hexose transport, and amino acid transport, measured by horseradish peroxidase, [14C]aminoisobutyric acid, and deoxy[3H]glucose uptake. alpha-Difluoromethylornithine also blocked the isoproterenol-induced increase in 45Ca influx and efflux and 45Ca redistribution; 0.5 mM putrescine nullified alpha-difluoromethylornithine inhibition and restored the increment in polyamines, 45Ca fluxes, endocytosis, hexose transport, and amino acid transport. These data implicate polyamine synthesis in isoproterenol stimulation of Ca2+ fluxes and membrane transport processes and support a model for signal transduction and stimulus-response coupling in which ornithine decarboxylase activation and polyamine synthesis play a pivotal role in regulating Ca2+ fluxes. In this model the polyamines generate local Ca2+ signals by stimulating Ca2+ influx or mobilizing intracellular calcium (or both) through a cation exchange reaction.

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.

We have characterized in Trypanosoma cruzi a DNA topoisomerase capable of decatenating complex trypanosomal kinetoplast DNA networks in the absence of ATP. The enzymatic activity requires Mg2+ and K+. Using a defined DNA topoisomer we showed that the linking number changes by steps of 2, which characterizes the enzyme as a type II topoisomerase. The enzyme can catenate supercoiled DNA molecules, unknot DNA, and cleave double-stranded DNA. The enzyme has no ATPase activity. The native enzyme has an Mr of about 200,000. Crude extracts and partially purified fractions contain an aggregating factor that can substitute spermidine in catenating reactions. Because of the presence of this factor, the kinetoplast DNA can only be decatenated by purified fractions. The enzyme is inhibited by certain drugs and provides a potential target for chemotherapy. Such an enzyme was also characterized in Trypanosoma equiperdum.

In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 microM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA. By Northern blot analysis three RNA transcripts were found to hybridize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximately 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.

Gy, along with Hyp, is a dominant mutation of the normal gene Pex causing X-linked hypophosphatemia in the mouse. Hemizygous Gy male mice, however, have greater defects in survival, bodily growth, skeletal mineralization, and neurological function than those found in heterozygous Gy females or in Hyp mice. Since the gene for spermine synthase is immediately upstream of the homologous human gene PEX, we compared the effects of the Gy and Hyp mutations on both the spermine synthase gene and the Pex gene. Barely detectable levels of spermine (< 5% of normal) with elevated levels of its precursor, spermidine, were found in organs of Gy male mice compared to normal male littermates. Neither Gy females nor Hyp male mice were significantly affected. Four missing introns of the spermine synthase gene were identified in Gy male mice, suggesting extensive gene disruption. A pseudogene for spermine synthase was also identified in the mouse genome. Pex mRNA was found in several but not all tissues studied in adult normal mice. Pex mRNA was altered in both Gy and Hyp mice. All male Hyp mice were lacking the 3' end of the Pex message, whereas all male Gy mice were deficient at the 5' end. In summary, the Gy mutation is associated with a recessively expressed mutation of the spermine synthase gene, leading to spermine deficiency, and a dominantly expressed mutation of the Pex gene, leading to hypophosphatemia. Alterations in two contiguous genes in Gy may explain the additional phenotypic abnormalities present in the Gy male mouse.

Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA.

Short catalytic RNAs with inherent, specific endoribonuclease activity, called ribozymes, have recently been shown to exist in nature. According to the structural models artificial ribozymes have been designed that can potentially hydrolyse any chosen target RNA sequence in trans at a specific site. We have constructed and characterized in vitro hammerhead and hairpin ribozymes designed to cleave viral RNA segment 5 of influenza A virus. Both ribozymes were functional under optimal in vitro conditions, but quantitative measurements indicate that the hammerhead ribozyme is considerably more efficient at this target site than the hairpin ribozyme. Mg2+ dependent hammerhead ribozyme-mediated cleavage reactions were enhanced at higher temperature and in presence of spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recombinant plasmids derived from transfection vector pSV2-neo were engineered to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids were introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selection of neomycin-resistance. Several permanent ribozyme-expressing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were determined and monitored by polymerase chain reactions. It was found that the highest levels (up to 70-80%) of resistance to influenza A virus strain X-31 super-infection was observed in COS cells transfected with plasmids containing SV40 early or SV40 early+late promoters coinciding with relatively high and constitutive rates of ribozyme expression. These results suggest the feasibility of developing ribozymes designed against influenza virus to achieve therapeutic value.

1. Direct or indirect inhibitors of l-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to l-ornithine, including dl-alpha-difluoromethylornithine, alpha-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-l-methionine decarboxylase activity (EC 4.1.1.50). 2. Comparison of the catalytic properties of S-adenosyl-l-methionine from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis-(guanylhydrazone). 3. Actinomycin D and cycloheximide, and RNA and a proteinsynthesis inhibitor respectively, blocked the increase of S-adenosyl-l-methionine decarboxylase activity elicited by alpha-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated S-adenosyl-l-methionine decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. The present results suggest that elevation of S-adenosyl-l-methionine decarboxylase activity by alpha-difluoromethylornithine is due to stabilization of the enzyme. 4. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased S-adenosyl-l-methionine decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and S-adenosyl-l-methionine decarboxylase activity could be established. 5. Addition of 1mm-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of S-adenosyl-l-methionine decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with alpha-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10mum) was as effective as spermidine in decreasing S-adenosyl-l-methionine decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. 6. The spermidine site of action appears to be post-translational since (a) the spermidine-induced decrease of S-adenosyl-l-methionine activity was not prevented by actinomycin D and (b) spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine and substantiate previous findings [Mamont, Duchesne, Grove & Tardif (1978) Exp. Cell Res.115, 387-393]. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of S-adenosyl-l-methionine decarboxylase is discussed.

THE SENESCENCE (PROTEOLYSIS AND LOSS OF CHLOROPHYLL) OF ISOLATED LEAVES OF OAT SEEDLINGS IN THE DARK IS INHIBITED OR DELAYED BY COMPOUNDS OF SIX DIFFERENT TYPES: phenazine methosulfate, fusicoccin, alpha,alpha'-dipyridyl, cycloheximide, spermidine, and two cytokinins. In every case but the last, these compounds in optimum concentration caused the stomata to open and remain partly or completely open throughout the 72- or 96-hr experimental period. The cytokinins caused only a partial opening, which is ascribed to their exerting two different effects. Taken together with the previous report that five different treatments that accelerated or promoted senescence in the light caused stomatal closure or occlusion, these data establish a general parallel between stomatal aperture and senescence, with strong indication that the stomatal aperture is the causal factor. A possible explanation of the relationship is proposed.

We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C. In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency. The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains.

Several polyamines have been shown to interact with a site on the N-methyl-D-aspartate (NMDA) receptor that regulates the binding of open channel blockers. Spermine (SP) and spermidine (SD), polyamine agonists, enhanced binding of open channel blockers, whereas arcaine (ARC), diethylenetriamine (DET), and putrescine (PUT), polyamine antagonists, reduced the polyamine enhancement of open channel blocker binding. We previously showed that SP had multiple actions on NMDA receptor single-channel currents that underlie its effect on whole-cell NMDA receptor current. At high concentrations, SP produced a voltage-dependent decrease in NMDA receptor single-channel conductance and average open time. In the present study, another polyamine agonist (SD) produced a similar reduction of NMDA receptor single-channel conductance at higher concentrations. The polyamine antagonists (ARC, DET, and PUT), however, produced a voltage-dependent reduction in NMDA receptor whole-cell currents and reductions in single-channel conductance and average open time, even in the absence of polyamine agonists. The rank order of potency for reduction of NMDA receptor single-channel conductance by polyamines was ARC greater than SP greater than SD greater than PUT = DET, a rank order similar to that for the inhibitory actions of polyamines in receptor binding assays and for the effects of the antagonists on NMDA receptor whole-cell currents. The polyamine antagonist DET did not block the reduction of single-channel conductance by the polyamine agonist SP. In fact, the effects of SP and DET on single-channel conductance were additive. DET also showed a variable enhancement of NMDA receptor whole-cell currents in some neurons, suggesting polyamine agonist-like properties. These results are not consistent with the standard pharmacological profile for agonists and antagonists acting at the same site. Potential mechanisms for the effects of the polyamines on single-channel conductance are discussed.

Putrescine, spermidine, and spermine can be covalently incorporated as the corresponding peptide-bound gamma-glutamyl-polyamine derivatives into selected glutaminyl residues in proteins and polypeptides that serve as amine acceptor substrates for various calcium-dependent transglutaminases. Once aliphatic diamines or polyamines have been enzymatically incorporated into polypeptides in that fashion, the remaining free primary amino groups can undergo further transglutaminase-catalyzed attachment to other reactive glutaminyl residues to yield bis-(gamma-glutamyl)polyamine cross-bridges. This essay considers mechanistic features of these reactions as catalyzed by various forms of transglutaminases present either extracellularly or intracellularly in mammals. The potential physiological significance of the reactions in mammalian cells and body fluids is discussed.

The uptake of polyamines, methylglyoxal bis(guanylhydrazone) (MGBG), and paraquat [N,N-dimethyl-4,4'-bipyridylium] into control Chinese hamster ovary (CHO) cells and a mutant CHO cell line selected for resistance to the toxicity of MGBG was examined. In contrast to control CHO cells, the mutant cells had no detectable uptake of MGBG or any of the polyamines. There was no difference between the two cell lines in the uptake of alpha-aminoisobutyric acid (AIB), which indicates that there was no general change in membrane transport processes. The mutant cells were also found to be resistant to the toxicity of paraquat and to have a reduced capability to take up the herbicide. This finding confirms that the uptake of paraquat is necessary for the toxicity of this compound and that the paraquat is taken up by a transport system that also transports MGBG. Competition experiments showed that an excess of unlabeled paraquat inhibited uptake of MGBG and, to a lesser extent, uptake of putrescine and spermidine, but no inhibitory action on spermine uptake could be detected. Studies with type II cells isolated from rat lung also demonstrated uptake of paraquat and spermidine, but paraquat was only a weak inhibitor of spermidine uptake in this system. These results suggest that there may be multiple systems for the uptake of MGBG and polyamines and that paraquat is taken up by at least one but not by all of these systems.

The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine>> spermidine>> Mg2+>> Na+>> Tris-HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.

Natural polyamines putrescine, spermidine and spermine are ubiquitous cellular components. Recent studies showed that these compounds are capable of provoking a conformational transition in poly(dG-m5dC).poly(dG-m5dC) from its usual right-handed B-DNA form to a left-handed Z-DNA form at physiologically relevant cationic concentrations. We studied the efficacy of spermidine, six homologs of spermidine (H2N(CH2)nNH(CH2)3NH2, where n = 2 to 8 (n = 4 for spermidine)) and diethylene triamine to provoke the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) using a monoclonal anti-Z-DNA antibody and spectroscopic techniques. The concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 30 +/- 1 microM. Chemical structural effects were significant when the spermidine homologs were used to induce the transition. The midpoint concentration increased as the number of -CH2 groups varied in relation to that of spermidine. We interpret these structural effects on the basis of molecular models of the interaction of polyamines with polynucleotides.

The association of N1-acetylspermidine with human colorectal adenocarcinomas has been evaluated in this study. Free polyamines and their monoacetylated forms in adenocarcinomas, adenomas, and apparently healthy mucosae were determined using high-performance ion-exchange chromatography. The N1-acetylspermidine levels in well- and moderately differentiated adenocarcinomas were 27.30 +/- 3.13 (S.E.) (n = 99) and 22.86 +/- 3.60 (n = 22) nmol/g, wet weight, respectively. These values were significantly higher than those of benign adenomas (5.38 +/- 0.85 nmol/g, n = 31) and of control mucosae. The N1-acetylspermidine levels in control mucosae on the oral and anal side of adenocarcinomas were 5.84 +/- 1.44 (n = 57) and 7.92 +/- 2.89 (n = 50) nmol/g, respectively; no significant difference was observed between control mucosae and adenomas. The mean levels of three polyamines, putrescine, spermidine, and spermine in both adenomas and adenocarcinomas were about twice as high as those of control mucosae. The molar ratios of spermidine to spermine were significantly greater in both adenomas and adenocarcinomas than in control tissues. There was no obvious correlation between the free polyamine concentrations and the degree of malignancy of the colorectal tumors. These results suggest that the metabolism of N1-acetylspermidine in colorectal adenocarcinomas is quite different from that in adenomas and in nonneoplastic mucosae and that N1-acetylspermidine can be a promising biochemical marker of cancer in the human large intestine.

The beta-adrenergic receptor kinase, which specifically phosphorylates the agonist-occupied beta-adrenergic receptor, is strongly inhibited by polyanions. Heparin and dextran sulfate inhibit the enzyme with an IC50 of approximately 0.15 microM. De-N-sulfated heparin is approximately 8-fold less potent. Other acid mucopolysaccharides such as heparan sulfate and chondroitin sulfates B and C are also less effective. Polyaspartic and polyglutamic acid also inhibit with IC50 values of 1.3-2 microM. Inositol hexasulfate, with an IC50 of 13 microM is approximately 270-fold more potent than inositol hexaphosphate implicating the sulfate group as a major determinant of the inhibition. The inhibition by heparin is competitive with substrate and of mixed type with respect to ATP. Polycations also inhibit receptor phosphorylation by beta-adrenergic receptor kinase. Polylysine is more effective with an IC50 of 69 microM, while spermine (990 microM) and spermidine (2570 microM) are less potent. Polylysine, spermine, and spermidine are also able to block effectively the inhibition by heparin. The identification of compounds which specifically inhibit beta-adrenergic receptor kinase should prove useful in further defining the biological role of this enzyme.

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.