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Results for “Spermidine”

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At least 19 recordsLinked to original sources

PubMed1991

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells.

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.

Affinity Labels↗

PubMed1991

Establishment and preliminary application of spermidine radioimmunoassay with 125I-labelled monoclonal antibody and solid phase antigen.

Purified anti-spermidine monoclonal antibody was labelled with radioactive iodine by the Iodogen method and spermidine-bovine serum albumin (SPD-BSA) conjugate was used to coat polystyrene beads as solid phase antigen. The new solid phase 125I-labelled spermidine radioimmunoassay (RIA) depends on the competition between spermidine in the sample and the solid phase antigen for the limited amount of 125I-labelled monoclonal antibody. The sensitivity of this assay was 10 ng/ml higher than that of liquid RIA for spermidine with 14C-labelled spermidine. The coefficients of variation (CV) within and among batches were 4% and 13% respectively. The sample-batch capacity was increased from 20 (liquid RIA with 14C-labelled spermidine) to 150-200 by using this method. Because of its simplicity, the solid phase RIA kit is very convenient for population survey. This RIA could be used to determine spermidine in saliva for the diagnosis of precancerous lesions. In a preliminary study saliva spermidine levels in different populations were measured among 130 normal subjects, 202 esophageal epithelial hyperplasia cases treated with anti-tumor B for 5 years, 207 esophageal epithelial hyperplasia cases as control, and 55 esophageal cancer patients. The levels were 1,795 +/- 1,481, 3,470 +/- 6,981, 9,753 +/- 17,641 and 18,090 +/- 21,509 ng/ml, respectively, with the saliva spermidine levels in precancerous and cancer patients being significantly higher than that of normal subjects (P less than 0.001); the level in patients treated with anti-tumor B was significantly lower than that of controls (P less than 0.001). This decreased saliva spermidine content was coincident with the 47.3% reduction of canceration rate seen in precancerous patients after a 5-year treatment with anti-tumor B.

Antibodies, Monoclonal↗

PubMed1990

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules.

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.

Binding Sites↗

PubMed1992

Spermidine nuclear acetylation in rat hepatocytes and in logarithmically growing rat hepatoma cells: comparison with histone acetylation.

Spermidine acetylation has been studied in nuclear homogenates and in entire nuclei from rat hepatocytes and rat hepatoma tissue culture (HTC) cells, isolated at different stages of logarithmic growth, and compared to histone acetylation. Under all experimental conditions, N8-acetylspermidine was the predominant product of the reaction (90%). Unlike histone, spermidine acetylation in HTC cell and hepatocyte entire nuclei was almost absent or strikingly reduced relative to acetylation using nuclear homogenates as the enzyme sources. This was due to the lack of a free minor pool of spermidine, most likely lost during the purification of entire nuclei. Thus, preincubation of intact nuclei in the presence of spermidine restored activities to values observed using nuclear sonicates. Spermidine acetylation in HTC cell nuclei fluctuated moderately during cell growth, being stimulated immediately after initiation of proliferation and decreasing progressively as cultures reached high cell density. This pattern corroborated that of N8-acetylspermidine intracellular accumulation induced by culturing cells in the presence of 1 mM 7-amino-2-heptanone, a competitive inhibitor of N8-acetylspermidine deacetylase. Histone acetylation during HTC cell growth was not markedly different qualitatively from that of spermidine. Moreover, spermidine and histone acetylations in hepatocyte nuclei were of the same order of magnitude as those seen in rat hepatoma cell nuclei. Finally, inhibition of deacetylation of N8-acetylspermidine had no apparent deleterious effects on cell and growth. It remains to be determined whether the acetylation step is of higher physiological importance, in particular, and as discussed in nuclear spermidine turnover.

Acetylation↗

PubMed1990

Spermidine enhancement of [3H]MK-801 binding to the NMDA receptor complex in human cortical membranes.

The effects of spermidine on the binding of [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor complex was studied in human cerebral cortical membranes. [3H]MK-801 binding was increased from 56 +/- 5 fmol/mg protein (mean +/- S.E.M., n = 7) to 319 +/- 71 fmol/mg protein in the presence of 200 microM spermidine. The ED50 for spermidine stimulation of [3H]MK-801 binding was 89 +/- 22 microM (mean +/- S.E.M., n = 6). In the presence of glutamate (1 microM) plus glycine (1 microM) the ED50 was reduced to 5.5 +/- 0.7 microM. The increase in binding in the presence of spermidine was characterised by an increase in the rate of association of [3H]MK-801. In the presence of spermidine. [3H]MK-801 was inhibited by AP5. 7-chlorokynurenic acid and ifenprodil with IC50 values of 0.5 +/- 0.3 24 +/- 19 and 91 +/- 28 microM, respectively. None of these antagonists was a competitive inhibitor of the spermidine stimulation of [3H]MK-801 binding. Thus spermidine modulates the NMDA receptor complex in human brain, providing further evidence that the complex is similar in rat and human cortex.

Adrenergic alpha-Antagonists↗

PubMed1978

Role of spermidine in casein gene expression in the rabbit.

Spermidine concentration in rabbit mammary gland was estimated during pregnancy, lactation and after the induction of milk synthesis by prolactin and glucocorticoids in vivo and in vitro. It was observed that mammogenesis and lactogenesis during preganancy and the initiation of milk secretion at parturition are accompanied by an enhancement of spermidine concentration in the mammary gland. By contrast, the initiation of these phenomena by hormone injections does not require such variations of spermidine concentration. In organ culture, a slight increase in spermidine concentration was obtained under the influence of an hormonal combination including insulin, prolactin and cortisol. Spermidine added to the culture medium was unable to mimic cortisol action. An amplification of casein synthesis and a parallel increase of casein mRNA concentration was provoked by cortisol even when spermidine synthesis was blocked. Thus, one of the major actions of glucocorticoids during lactogenesis in the rabbit is not mediated through an increase in spermidine concentration in the mammary gland.

Animals↗

PubMed1978

Polyamines of Anacystis nidulans and metabolism of exogenous spermidine and spermine.

Several biochemical parameters, including that of polyamine content, accompanying the growth of the cyanobacterium Anacystis nidulans were studied. At all stages of growth under autotrophic conditions, the organisms were found to be rich in spermidine and lacking in spermine, as is typical of procaryotic organisms. The cells were quite low in putrescine, and no unusual polyamine was observed to be present as a major component. Conjugated polyamines were not detected in the cultures. At maximal culture density, the levels of spermidine, DNA, RNA, protein, and chlorophyll were also maximal. Shortly after the inception of the stationary phase, the spermidine content of the cells was the first parameter observed to decrease in cultures which were shortly to become yellow. Spermidine lost from the cells was not recovered in the medium in a free or conjugated form. This indication of degradation of spermidine was studied by the addition of polyamines to growing cultures. Exogenous spermidine and spermine were found to be metabolized rapidly by the organisms, of which diaminopropane was one product. Putrescine was found to be markedly toxic, whereas spermidine, some other triamines, and spermine were much less toxic.

Chlorophyll↗

PubMed1978

Effect of spermidine on bacteriophage P22 infection.

The effect of spermidine on phage P22 infection of Salmonella typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infective process, the replication of the phage was inhibited but the cells did not survive. If spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.

Biological Transport↗

PubMed1991

Role of unsaturated derivatives of spermidine as substrates for spermine synthase and in supporting growth of SV-3T3 cells.

Synthetic unsaturated analogues of the natural polyamine were examined as possible substrates for spermine synthase and as replacements for spermidine in supporting the growth of SV-3T3 cells. It was found that N-(3-aminopropyl)-1,4-diamino-cis-but-2-ene [the cis isomer of the alkene analogue of spermidine] was a good substrate for spermine synthase, but that the trans isomer [N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and the alkene analogue [N-(3-aminopropyl)-1,4-diaminobut-2-yne] were not substrates. These results provide the first demonstration of stereospecificity in the spermine synthase reaction. All three of the unsaturated spermidine analogues described above and the cis-alkene analogue of spermine [N1N4-bis-(3-aminopropyl)-1,4-diamino-cis-but-2-ene] were able to support the growth of SV-3T3 cells that were prevented from the endogenous synthesis of spermidine by treatment with alpha-difluoromethylornithine. Since N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and N-(3-aminopropyl)-1,4-diaminobut-2-yne were not converted into a spermine derivative, it is apparent that this conversion is not needed for the stimulation of growth. However, since N1N4-bis-(3-aminopropyl)-1,4-diamino-cis-but-2-ene was also able to support growth and was not degraded to the spermidine derivative, it appears that either polyamine can be effective in this respect. All of the unsaturated analogues tested accumulated in the SV-3T3 cells to a much greater extent than spermidine itself. This indicates that these compounds are substrates for the polyamine transport system, but that they are less effective than the natural polyamines in the feedback regulation of this system.

Animals↗

PubMed1977

Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli.

The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 10(5) M-1 in 1 mM sodium cacodylate, pH 7.5, to 3 x 10(2) M-1 in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.

Binding Sites↗

PubMed1977

Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2.

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.

Bacterial Proteins↗

PubMed1992

Erythrocyte spermidine levels in IDDM patients.

OBJECTIVES: To evaluate whether erythrocyte levels of polyamines spermidine and spermine (expressed in nmol/ml packed erythrocytes [PRBCs]) are modified in insulin-dependent diabetes mellitus (IDDM) and are associated with the presence of retinopathy or nephropathy. RESEARCH DESIGN AND METHODS: We studied erythrocyte spermidine and spermine levels in 38 IDDM patients with or without persistent microalbuminuria (urinary albumin excretion rate [AER] between 20 and 200 micrograms/min), macroalbuminuria (AER greater than 200 micrograms/min), or retinopathy compared with 60 sex- and age-matched control subjects. RESULTS: Mean +/- SD erythrocyte spermine content was similar in both diabetic (9.7 +/- 5.5 nmol/ml PRBCs) and control (8.8 +/- 3.5 nmol/ml PRBCs) subjects, whereas spermidine was higher in diabetic (19.1 +/- 7.2 nmol/ml PRBCs) than in control (14.5 +/- 4 nmol/ml PRBCs, P = 0.0007) subjects. Moreover, spermidine was significantly higher in the groups with microalbuminuria (n = 11, 22.5 +/- 9.2 nmol/ml PRBCs) and macroalbuminuria (n = 4, 22.2 +/- 5.7 nmol/ml PRBCs) than in both normoalbuminuric (n = 23, 16.9 +/- 5.6 nmol/ml PRBCs) and control (F = 9.78, P = 0.0001) subjects, and correlated with log AER (r = 0.41, P = 0.009). Similarly, proliferative retinopathy was associated with a significant increase in spermidine (n = 5, 20 +/- 7 nmol/ml PRBCs compared with control subjects [P = 0.0009]). CONCLUSIONS: Our data suggest that erythrocyte spermidine content is increased in IDDM patients associated with both diabetic nephropathy and advanced retinopathy.

Adult↗

PubMed1992

Interactions between zinc and spermidine on the N-methyl-D-aspartate receptor complex: clues to the mechanism of action of 1,10-bis(guanidino)decane and pentamidine.

This study investigated the mechanism of action of two novel, noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor using [3H]dizocilpine binding to rat brain membranes. Pentamidine and 1,10(bisguanidino)decane (BG10) inhibited [3H]dizocilpine binding in the concentration range of 1 to 100 microM, as did Zn++ and the competitive polyamine antagonist arcaine. The action of each of these agents was sensitive to the addition of spermidine to the assay. However, only arcaine interacted with spermidine in a competitive fashion. Spermidine decreased the apparent affinity of Zn++ and also increased the Hill slope of the Zn++ inhibition curves. BG10 and pentamidine inhibition of [3H]dizocilpine binding was less sensitive to spermidine. Calcium had similar effects to spermidine on the inhibition of [3H]dizocilpine binding by Zn++, arcaine, pentamidine and GB10, but was less potent than spermidine. Treating membranes with diethylpyrocarbonate, a histidine-modifying reagent, decreased the apparent affinity of Zn++ by more than 4-fold while having very modest effects on the actions of BG10, pentamidine and arcaine. In addition, Zn++ failed to slow the dissociation of [3H]dizocilpine in diethylpyrocarbonate-treated tissue, whereas the action of BG10 and pentamidine was qualitatively unaffected. These data show that the effects of BG10 and pentamidine on the NMDA receptor are complex and may involve more than one binding site for each drug. In addition, this study shows that the action of Zn++ on the NMDA receptor is modulated by polyamines. Finally, the mechanism of action of pentamidine and BG10 cannot be attributed to an action at the Zn++ recognition site.

Animals↗

PubMed1976

Changes in serum putrescine and spermidine levels following local radiation to hepatoma 3924A of the rat.

Local irradiation of tumors of rats bearing 3924A hepatomas resulted in more than a doubling of the putrescine levvel and nearly a doubling of spermidine concentration in the serum within 12 hr. Within 24 hr, the putrescine concentration had increased four-fold in the serum, along with a continued increase in the spermidine concentration. The decrease in the spermidine concentration of the tumor paralleled increased levels of spermidine in the serum, whereas the concentrations of polyamines in the liver were unchanged. These changes are similar to previously reported changes in the spermidine concentration in sera and in tumors following the administration of a single dose of 5-fluorouracil to rats with 3924A hepatomas. Since local irradiation was confined to the tumor, we conclude that the increases in putrescine and spermidine detected in the serum are derived from the tumor tissue with no involvement of the host tissues.

Animals↗

PubMed1990

Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD.

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.

Animals↗

PubMed1990

Influence of the 5'-untranslated region of ornithine decarboxylase mRNA and spermidine on ornithine decarboxylase synthesis.

The effect of the 5'-untranslated region of ornithine decarboxylase (ODC) mRNA and of spermidine on the translation of ODC mRNA was studied in a rabbit reticulocyte cell-free system. The ODC mRNAs, possessing different sizes of the 5'-untranslated region and named mODC241, mODC188, mODC85, and mODC24 according to the number of nucleotides in the 5'-untranslated region, were synthesized by in vitro transcription techniques through the use of plasmids containing various sizes of mouse ODC cDNA. As the size of the 5'-untranslated region of ODC mRNA increased, the efficiency of the translation decreased and the degree of stimulation of the translation by 0.2 mM spermidine increased. The inhibition of ODC mRNA translation by high concentrations of spermidine (0.6-1 mM) also increased with an increase in the size of the 5'-untranslated region. The spermidine effects were increased greatly when the size of the 5'-untranslated region of ODC mRNA was increased from 85 to 188 nucleotides. These results suggest that the nucleotides 70-220 upstream from the initiator AUG are involved in the decrease in translational efficiency. In addition, at least the nucleotides in the 70-170 region upstream from the initiator AUG are important in the strong stimulation of ODC synthesis by low spermidine concentrations and in the inhibition of ODC synthesis at high spermidine concentrations.

Animals↗

PubMedDate not supplied

[Formation of the compact form of DNA in solution after reaction with spermidine].

The formation of compact particles from double-stranded DNA molecules in water-salt solutions containing spermidine was studied. It has been shown that in solutions of low ionic strength (0.01 M NaCl) DNA-spermidine complexes have the form of large particles which scatter UV-light. Electron micrographs show that such complexes formed at certain molar ratios spermidine/DNA may exist both as intermolecular aggregates and as toroidal particles 1500 A in diameter. The CD spectra of solutions containing DNA-spermidine complexes are characterized by the positive band (delta epsilon max = 10) at 265--270 nm. The appearance of the positive CD band may be caused by two factors: interaction between DNA and spermidine may lead to the alteration of the DNA secondary structure "in direction to A-form" or intermolecular aggregation, which may change the initial shape of the CD spectrum. The exclusion of spermidine molecules from DNA-spermidine complexes by Na+ ions in presence of poly(ethylene glycol) which occurs as the ionic strength increases from 0.01 to 0.3 does not lead to decompactization of DNA molecules but is accompained by the appearance of the intense negative CD band at 270 nm.

Chemical Phenomena↗

PubMed1979

The synthesis and turnover of spermidine and spermine in mouse brain.

Following the administration to mice of radiolabeled putrescine by intraventricular injection, changes in the specific radioactivity of putrescine, spermidine, and spermine have been measured. Putrescine decline was biphasic, being more rapid over the first 12 hr(t 1/2 = 5 hr) than over the remainder of the 48-hr period (t 1/2 = 11 hr) that significant labeling was detected. Spermidine was rapidly labeled during the decline in putrescine radioactivity and maximum incorporation of label occurred at 18 hr. Subsequently, spermidine specific activity declined with a half-life of 22 days. Spermine synthesis was slower, with maximum labeling occurring after 4 days. Spermine turnover, measured at a time when spermidine radioactivity had substantially declined, was extremely slow (t 1/2 = 92 days). The data supports the view that putrescine is a precursor of spermidine which in turn is required for spermine synthesis.

Animals↗