The determination of N1-acetylspermine in mouse liver

N¹-Acetylspermine has been postulated to be an intermediate in the conversion of spermine to spermidine. This compound, together with N¹-acetylspermidine has now been detected in the liver of mice which were pretreated with tetrachloromethane. The following methods were used for the identification of N¹-acetylspermine: (a) High-pressure liquid-chromatography of the non-derivatized amines on a reversed-phase column, using octane sulfonate for ion-pairing. (b) Thin-layer chromatography of the dansyl derivatives. (c) Mass spectrometry of the dansyl derivatives. Both chromatographic methods allowed the quantitative estimation of N¹-acetylspermine and N¹-acetylspermidine in the liver of tetrachloromethane-treated animals.     

Deacetylation of N8-acetylspermidine by subcellular fractions of rat tissue

The in vitro deacetylation of N⁸-acetylspermidine by an enzyme activity in rat tissues is described. This deacetylase activity occurs as a soluble, cytoplasmic enzyme in rat liver and was detected in the 100,000g supernatant fraction of all tissues examined. The highest specific activity was found in liver. Spleen, kidney, and lung were found to contain 20–50% of the activity in liver, while heart, brain, and skeletal muscle exhibited from 2 to 10% of the activity in liver. Serum contained only barely detectable levels of activity, much lower than any of the tissues studied. The in vitro metabolism of N¹-acetylspermidine differed from that observed for N⁸-acetylspermidine and does not appear to involve a simple deacetylation reaction.     

Effect of carbon tetrachloride on polyamine metabolism in rodent liver

Administration of hepatotoxic doses of carbon tetrachloride to mice produced a 25-fold increase in spermidine/spermine N¹-acetyltransferase activity within 6 h, but did not significantly change the activity of polyamine oxidase. The content of acetylated polyamines in the mouse liver was increased more than 100-fold from levels below the limit of detection to 0.6 μmol of N¹-acetylspermidine and 0.045 μmol of N¹-acetylspermine per gram of tissue. Putrescine levels also rose by 7-fold within 6 h and by 21-fold within 24 h. These results are in contrast to changes in hepatic polyamines brought about in the rat by carbon tetrachloride. Although the hepatotoxin produced a similar increase in spermidine/spermine N¹-acetyltransferase in this species, the rise in acetylated polyamines was much smaller and more transient. The content of N¹-acetylspermidine was increased only to 0.066 μmol/g and N¹-acetylspermine was not detected. However, in the rat putrescine increased 35-fold within 6 h and 64-fold by 16 h. These differences appear to be due to the much higher polyamine oxidase activity which was 20 times greater in the rat than in the mouse liver. This oxidase converts N¹-acetylspermine to spermidine and degrades N¹-acetylspermidine to putrescine. Spermine content was significantly reduced in both species after exposure to carbon tetrachloride, but only part of this decline could be attributed to the increased acetylation.     

Formation of N1-acetylspermidine in rat liver after treatment with carbon tetrachloride

A single intraperitoneal injection of carbon tetrachloride produced a significant increase in the concentration of N¹-acetylspermidine in rat liver. The concentration of N¹-acetylspermidine was maximal at the same time after injection at which other workers reported maximal conversion of spermidine to putrescine and maximal acetylase activity in liv liver extracts. N¹-acetylspermidine was not detectable in livers of untreated animals and at 45 hours after injection with monoacetylation of polyamines precedes their degradation by polyamine oxidases. Spleen, lungs and erythrocytes of untreated animals contained detectable amounts of the monoacetyl polyamines. Treatment with carbon tetrachloride did not produce changes in the concentrations of the monoacetyl polyamines in these tissues.     

Acetylated polyamines as substrates for human pregnancy serum diamine oxidase

Human pregnancy serum diamine oxidase was purified 50 fold and tested for activity with a variety of substrates. Putrescine, spermidine, spermine, N-acetylputrescine, N⁸-acetylspermidine, and N¹-acetylspermidine were acceptable substrates for the enzyme, which exhibited greatest activity against N¹-acetylspermidine.     

Acetylputrescine deacetylase from Micrococcus luteus K-11

An acetylputrescine deacetylase was induced in Micrococcus luteus K-11, and was partially purified and characterized briefly. The enzyme was most active toward acetylputrescine, followed by N⁸-acetylspermidine and acetylcadaverine, but was inactive toward N¹-acetylspermidine and N¹-acetylspermine. The K_m value for acetylputrescine was 0.321 mM. It was almost unaffected by -SH blocking agents but was inhibited by metal ions such as Cu²⁺ and Ni²⁺. Its molecular weight estimated by Sephacryl S-200 column chromatography was 115000.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

Conversion of exogenous spermidine into putrescine after administration to rats

Administration of large, but non-toxic doses of spermidine (0.4–1.25 mmol/kg) led to a substantial increase in putrescine in liver, kidney and a number of other tissues including muscle. The increase in putriscine peaked at 6 h after treatment and was completely prevented by administration of cycloheximide 3 h after the spermidine suggesting that the induction of a new protein was required. This protein is likely to be spermidine N¹-acetyltransferase which was induced by the treatment with spermidine and increased 3–4-fold in liver and kidney within 6 h. N¹-Acetylspermidine was detected in tissues at this time after spermidine treatment and experiments in which labeled spermidine was given indicated that a substantial fraction of the administered spermidine was converted into N¹-acetylspermidine and into putrescine. These results suggest that the rise in putrescine after spermidine treatment is brought about by the production of N¹-acetylspermidine which is converted into putrescine by the action of polyamine oxidase. The limiting step in this conversion is the activity of the acetylase which is induced in response to the rise in spermidine content. The acetylase/oxidase pathway, therefore, provides a means by which polyamine levels can be regulated and excess polyamine disposed of.     

Occurrence of sym-homospermidine in extremely thermophilic bacteria

Triamines produced by an extreme thermophile, Thermus thermophilus, were isolated and their chemical structures were determined. It was found that two novel triamines, norspermidine (1,7-diamino-4-azaheptane, NH₂(CH₂)₃· NH(CH₂)₃NH₂) and sym-homospermidine (1,9-diamino-5-azanonane, NH₂(CH₂)₄NH· (CH₂)₄NH₂) are present in the thermophile cells in addition to spermidine (1,8-diamino-4-azaoctane, NH₂(CH₂)₃NH(CH₂)₄NH₂).     

Pontibacter jeungdoensis sp. nov., isolated from a solar saltern in Korea

A Gram-staining-negative, rod-shaped and red-pigmented bacterial strain, HMD3125^T, was isolated from a solar saltern in Jeungdo, Republic of Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMD3125^T formed a lineage within the genus Pontibacter and was similar to Pontibacter salisaro (96.1%) and P. korlensis (95.3%). The major fatty acids of strain HMD3125^T were summed feature 4 (comprising iso-C_17:1 I and/or anteiso-C_17:1 B; 30.4%), iso-C_15:0 (20.4%) and iso-C_17:0 3OH (17.2%). The polar lipid profile of HMD3125^T consisted of the phosphatidylethanolamine, four unidentified polar lipids, unidentified phospholipid, unidentified aminolipid and unidentified aminophospholipid. Strain HMD3125^T contained MK-7 as the predominant menaquinone and sym-homospermidine as the major polyamine. The DNA G+C content of strain HMD3125^T was 45.6 mol%. Strain HMD3125^T assigned as a novel species in the genus Pontibacter, for which the name Pontibacter jeungdoensis sp. nov. is proposed. The type strain is HMD3125^T (=KCTC 23156^T =CECT 7710^T).     

Secretin-induced accumulation of N1-acetylspermidine and putrescine in the rat pancreas

The effects of secretin on polyamine metabolism in rat pancréas were investigated. Single injections of secretin increased ornithine decarboxylase activity only very slightly. However a substantial time- and dose-dependent increase of acetyl CoA: polyamine N¹-acetyltransferase activity was observed. The concentrations of N¹-acetylspermidine, putrescine and β-alanine increased concomitantly, but spermidine and spermine remained unchanged. These results suggest that, in this model, the accumulated putrescine was formed from spermidine, via its acetylation, rather than from ornithine.     

Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle

The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20°C. If inhibitors of ethylene formation or action (Co²⁺, aminoethoxyvinylglycine, or Ag⁺) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag⁺ is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag⁺ for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle.     

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry

A method for the quantification of acetylpolyamines, N¹,N¹²-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N¹,N⁸-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using ¹⁵N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of α,ω-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N¹-acetylspermidine and N⁸-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).     

Acetylation of spermidine in polyamine catabolism

Treatment with thioacetamide (150 mg/kg)_ was used to enhance polyamine metabolism in rat liver. The increased uptake and catabolism of [¹⁴C]spermine and the changes of putrescine, spermidine and spermine concentrations indicated enhanced polyamine turnover rates. The increase of hepatic putrescine concentration was accompanied by an increase of monoacetylputrescine and N¹-monoacetylspermidine concentration. In control animals, the latter compound was below detection levels. Thioacetamide treatment also enhanced putrescine excretion, which again was concomitant with an increased excretion of N¹-acetylspermidine.The close time-dependent correlation between induced putrescine formation and enhanced formation of N¹-acetylsperimidine at a time when liver spermidine and spermine concentrations are not changed, favors the notion that acetylation is an essential step in polyamine degradation and elimination. The increase of polyamine oxidase and decrease of acetylpolyamine deacetylase activities in the liver of thioacetamide-treated rats is in line with an increased polyamine turnover, but these enzymes. although essential, are not rate-limiting in the catabolic reactions.     

Cruoricaptor ignavus gen. nov., sp. nov., a novel bacterium of the family Flavobacteriaceae isolated from blood culture of a man with bacteraemia

A Gram-reaction-negative bacterium, strain IMMIB L-12475^T, was isolated from blood cultures of a human with septicaemia. The yellowish orange pigmented strain contained flexirubin pigment. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain IMMIB L-12475^T belonged to the family Flavobacteriaceae, forming a distinct phyletic line that is distantly related (79.1–89.4% sequence similarity) to described genera of this family. Membership to the family was confirmed by a fatty acid profile consisting of branched-chain and 3-hydroxy fatty acids with major amounts of iso-C_17:0 3-OH and iso-C_15:0, by the presence of menaquinone MK-6 as the only respiratory quinone and a polyamine pattern that contained sym-homospermidine as major component. The phospholipids consisted of phosphatidylethanolamine and an unknown phospholipid. The genomic DNA mol% G+C content was 45.6%. The distant phylogenetic position as compared to other representative of the family and the significant phenotypic properties such as pigment composition, morphology, and physiology support the proposal of a novel genus and species Cruoricaptor ignavus gen. nov., sp. nov. The type strain is IMMIB L-12475^T (=DSM 25479^T = CCUG 62025^T).     

Influence of dissimilarities in temporal and spatial N-uptake patterns on15N-based estimates of fixation and transfer of N in ryegrass-clover mixtures

The temporal N-uptake patterns of white clover (Trifolium repens L.) mixed with perennial ryegrass (Lolium perenne L.) and of red clover (Trifolium pratense L.) mixed with Italian ryegrass (Lolium multiflorum Lam.) were determined in successive harvests of herbage within the growth cycles of a ley established near Zürich (Switzerland). Rooting patterns were examined by injecting¹⁵N-fertilizer at soil depths ranging from 10 to 40 cm. The results were analyzed to determine the effect of variations in time and depth of N-uptake on the¹⁵N-based measurement of N from symbiosis (N_sym) and N from transfer (N_trans).Grasses in mixture appeared to have deeper rooting systems than grass monocultures, which led to an overestimation of N transfer from white clover to perennial ryegrass if¹⁵N was spread on the soil surface.White clover generally lagged behind grass in soil N- uptake. Soil N-uptake of red clover slowed down before that of the grass because % N_sym almost reached 100% during the second half of each growth cycle. However, the effect of these dissimilarities on the seasonal average of %N_sym did not exceed 2%.It is concluded that at the observed high levels of N₂ fixation, failure to account for the N-uptake patterns of the test and reference crops only slightly affected the estimates of % N_sym and % N_trans, and did not invalidate the observed differences between species.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine decarboxylase activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N¹-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N¹-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N¹-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N¹-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

Synthesis and antimicrobial activity of β-carboline derivatives with N2-alkyl modifications

β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure–activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N²-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N²-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01–0.05?μmol/mL. Our work highlights that N¹-N¹ dimerization and N²-benzylation significantly enhanced the antimicrobial effects of compounds.     

Biosynthesis of polyamines in Euglena gracilis

In Euglena gracilis Z the biosynthesis of spermidine and spermine closely resembles the pathways occurring in mammalian tissues and in most microorganisms. l-Ornithine and not l-arginine, as is the case in most plants, is the main precursor of putrescine, and S-adenosylmethionine donates the propylamino moiety for the biosynthesis of spermidine and spermine. Cell-free extracts of Euglena synthesized sym-norspermidine and sym-norspermine from 1,3-diaminopropane and labelled S-adenosylmenthionine. The synthases for the biosynthesis of these two polyamines have a pH optimum of 7.6, like that of spermidine and spermine synthases. Ion exchange chromatography showed two peaks corresponding to the retention times of 2,4-diaminobutyric acid and 1,3-diaminopropane, lower homologues of ornithine and putrescine, respectively. Experiments with dl-2,4-diaminobutyric acid-[4-¹⁴C] did not result in significant incorporation of the label into 1,3-diaminopropane.     

Functional homogeneity of P-700 in cyclic and non-cyclic electron transport reactions in thylakoids

The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP⁺ as acceptor, the illuminated steady-state concentration of P-700⁺ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo.The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg²⁺ or Ca²⁺, had no effect on the signal amplitude during the cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700⁺ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg²⁺. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.