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N Seiler

Publications and source records attributed to N Seiler.

At least 91 records · Page 5Linked to original sources
PubMed1989

New substrates of polyamine oxidase. Dealkylation of N-alkyl-alpha, omega-diamines.

1) N-Alkyl-alpha, omega-diaminoalkanes are substrates of polyamine oxidase. 2) The compounds are oxidatively cleaved and form equimolar amounts of an aldehyde, a diamine, and H2O2. 3) Minimum structural requirements of a substrate of polyamine oxidase are two positively charged amino groups and an alkyl-substituent on one or both nitrogen atoms. 4) Dealkylation of N-alkyl derivatives by polyamine oxidase in vivo is a method to accumulate diamines in brain, and to release intracellularly an aldehyde from a stable prodrug.

Animals↗
PubMed1989

On the subcellular localization of the polyamines.

Putrescine, spermidine and spermine were determined in the nuclear fraction of rat liver which was obtained by density gradient centrifugation in non-aqueous media, i.e. under conditions which avoid migration of water-soluble compounds. Calculations of the distribution of the polyamines between nuclear and extranuclear compartments were based on the assumption that the DNA is concentrated in the nuclei. No significant losses of the polyamines occurred during fractionation. From the polyamine determination in tissue and nuclear fraction it appeared that 16-17% of the liver spermidine and spermine, and about 8% of the putrescine content was localized in the nuclei. The spermidine/spermine-ratios in nuclei and whole tissue were not significantly different. Pretreatment of the animals with inhibitors of ornithine decarboxylase caused a decrease of putrescine exclusively in the extranuclear compartments, in agreement with a higher proportion of the inhibitors in the cytoplasm. Since the nuclear volume of rat liver corresponds to about 5% of total liver volume, the concentration of spermidine and spermine is higher in the nucleus than in extranuclear compartments. Published histochemical localizations of the polyamines suggested very low polyamine concentrations in the nuclei of non-dividing liver and HeLa cells, but dramatic polyamine accumulations in metaphase and anaphase nuclei. These results are in disagreement with previously reported autoradiographic data, subcellular localizations based on density gradient centrifugations, and with our present results. Since subcellular localization is a key issue in all attempts to clarify cellular functions of the polyamines the careful revision of the techniques involved in subcellular polyamine localizations seems imperative.

Adenosylmethionine Decarboxylase↗
PubMed1988

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine.

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.

Acetyltransferases↗
PubMed1988

5-Fluoromethylornithine, an irreversible and specific inhibitor of L-ornithine:2-oxo-acid aminotransferase.

5-Fluoromethylornithine (5-FMOrn) is the first specific irreversible inhibitor of L-ornithine:2-oxoacid aminotransferase (OAT) found. Single doses (greater than 10 mg/kg) of this compound inactivate OAT to a residual OAT-like activity. This activity (10-20% of total activity) is resistant to further inactivation by higher or repeated doses of 5-FMOrn, or incubation with the inactivator in vitro. Ornithine concentrations are greatly enhanced in various tissues, and urinary ornithine is dramatically increased, but no other amino acid is affected after acute treatment with 5-FMOrn. Repeated administration decreases carnosine and homocarnosine concentrations in brain. Toxic effects were not observed. The new inactivator is considered as a tool in the establishment of functions of OAT under physiological and pathological conditions.

Animals↗
PubMed1988

Chain-fluorinated polyamines as tumour markers. III. Determination of geminal difluoropolyamines and their precursor 2,2-difluoroputrescine in normal tissues and experimental tumours by in vitro and in vivo 19F NMR spectroscopy.

Treatment of laboratory animals bearing transplantable tumours with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, followed by 2,2-difluoroputrescine allows the partial replacement of the endogenous polyamines spermidine and spermine by their fluorine-containing analogs 6,6-difluorospermidine and 6,6-difluorospermine. Quantitative determination of the chain-fluorinated polyamines in excised tumor tissue by in vitro 19F NMR spectroscopy at 11.7 T gave results similar to those obtained by extraction and HPLC analysis. The NMR methods allowed the observation of several as yet unidentified geminal difluoropolyamine metabolites with a detection threshold as low as 1 nmole/g. Using a surface coil at 2.4 T, it was possible to detect in 10-20 min fluorinated polyamines in tumour, liver and bladder (urine) of mice in vivo at concentrations of the order of 50-100 nmol/g, i.e., when 2-5% of the natural polyamines have been replaced by their fluoro analogs. Since 6,6-difluorospermidine and 6,6-difluorospermine preferentially accumulate in tumours and specific tissues with enhanced polyamine metabolism, it is expected that these compounds may serve as probes for the application of 19F NMR methods for tumour diagnosis and therapy control. Since significant quantities of additional fluorine-containing polyamine metabolites were detected in some tissues, 19F NMR spectroscopy will contribute to our knowledge of polyamine metabolism in normal and tumour tissue.

Animals↗
PubMed1988

Chain-fluorinated polyamines as tumor markers. II. Metabolic aspects in normal tissues.

The objective of this work was to study certain metabolic aspects of fluorine-substituted analogues of natural polyamines in healthy experimental animals, with the aim of exploring their potential application as tumor markers. Tissue polyamine concentrations were more effectively depleted by combined treatment with D,L-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, and N1,N4-bis-allenylputrescine, an inactivator of polyamine oxidase, than with either inhibitor alone. This suggests the general importance of polyamine interconversion as a metabolic source of putrescine. Administration of 2,2-difluoroputrescine after 2 weeks pretreatment with the two inhibitors caused the formation of 6,6-difluorospermidine and 6,6-difluorospermidine in nearly all tissues. Highest concentrations of the chain-fluorinated polyamines were observed in the small intestine. At 24 h after 2,2-difluoroputrescine administration the amount was about 8% of the normal endogenous polyamine pool in the small intestine, but lower in all other tissues. Replenishment of endogenous polyamine pools is a relatively slow process. Approximately 9 days after cessation of treatment with the two inhibitors normal values had been reestablished. The rate of formation of endogenous polyamines was not affected by the presence of their difluoro analogues. Elimination of the chain-fluorinated polyamines from tissues seems not to follow normal polyamine metabolic patterns. Their most rapid elimination coincides with the enhancement of endogenous polyamines, indicating that the fluoro analogues are displaced by the natural polyamines. Most of the 2,2-difluoroputrescine was rapidly excreted in the urine, and formation of a conjugate was detected. 6,6-Difluorospermidine was also a urinary excretion product. However, the metabolic fate of 6,6-difluorospermine could not be clarified. It was not found in urine, either free or as conjugate. The relatively low accumulation of chain-fluorinated polyamines, together with their rapid elimination from normal tissues are characteristics which together with their previously established selective uptake into rapidly proliferating tissues recommend them as potential tumor markers that can be determined by 19F-NMR spectroscopy.

Animals↗
PubMed1988

Interrelationships between ornithine, glutamate, and GABA. II. Consequences of inhibition of GABA-T and ornithine aminotransferase in brain.

The objective of the present study was to compare the effects of elevation of GABA concentration and those of inactivation of L-ornithine: 2-oxoacid aminotransferase (OAT) on the in vivo metabolism of L-ornithine (Orn) in brain. Vigabatrin (4-aminohex-5-enoic acid) and gabaculine (5-amino-1,3-cyclohexadienyl carboxylic acid), two well known inactivators of GABA-T, were used to elevate brain GABA concentrations. The latter inactivates OAT also. Transamination of Orn is, from a quantitative point of view, a significant reaction in mouse brain. GABA is a feed-back regulator of OAT. Within GABAergic neurons Orn concentration may be regulated by endogenous GABA. Extensive inactivation of OAT causes a considerable increase of Orn concentration, both in synaptosomes and in non-synaptosomal compartments. The results are compatible with a role of Orn as precursor of glutamate and/or GABA in certain neurons.

4-Aminobutyrate Transaminase↗
PubMed1988

Chain-fluorinated polyamines as tumor markers--IV. Comparison of 2-fluoroputrescine and 2,2-difluoroputrescine as substrates of spermidine synthase in vitro and in vivo.

1. 2-Fluoroputrescine has a high affinity for spermidine synthase (Km 12 microM) and obeys normal Michaelis-Menten kinetics. 2. The only product of the spermidine synthase-catalysed aminopropylation of 2-fluoroputrescine is 6-fluorospermidine. Formation of the isomeric 7-fluorospermidine could not be detected. 3. 2,2-Difluoroputrescine has even a higher affinity for spermidine synthase than putrescine and 2-fluoroputrescine; however, at concentrations greater than 25 microM one observes inhibition of the aminopropylation reaction. 4. Competition experiments between putrescine and 2,2-difluoroputrescine revealed mixed type inhibition. 5. HTC cells in suspension culture incorporated only small amounts of 2-fluoroputrescine, and even less in the case of 2,2-difluoroputrescine, if they were exposed to 10 microM concentrations of these diamines for up to 24 hr. However, in the presence of 0.5 mM DFMO, a concentration not sufficient to decrease cell growth significantly, but sufficient to decrease cellular putrescine and spermidine concentrations, the uptake of the chain-fluorinated diamines and their transformation into the fluorinated polyamine analogues was dramatically enhanced. In comparison with the difluoro analogues the accumulation rate of monofluoropolyamines was greater by a factor of about two. 6. 6-Fluorospermidine and 6-fluorospermine could be detected in significant quantities in nearly all tissues of mice 48 hr after a single dose (500 mg/kg) of 2-fluoroputrescine. In an analogous experiment with 2,2-difluoroputrescine, the formation of chain-fluorinated polyamines was considerably smaller. 7. Pretreatment of Lewis lung carcinoma bearing C57BL mice with alpha-difluoromethylornithine enhanced the incorporation of 2-fluoroputrescine into all organs, except the brain. Tumor and small intestines showed by far the highest accumulation of 6-fluoropolyamines. 8. Under identical experimental conditions the accumulation of chain-fluorinated polyamines in tumor tissue was more than twice as high with 2-fluoroputrescine as precursor than with the same dose of 2,2-difluoroputrescine. In normal tissues the difference between the uptake of 2-fluoroputrescine and 2,2-difluoroputrescine was usually even greater. 9. From the fact that the accumulation of 6-fluoropolyamines is less selective in tumors than that of 6,6-difluoropolyamines, and from the lower detection sensitivity due to its lower fluorine content, we conclude that 2,2-difluoroputrescine is more advantageous as a tumor marker than 2-fluoroputrescine for detection with 19F-NMR spectroscopy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Synapse formation and development of neurotransmitter functions in neuronal cells from chick brain cultured in a serum-free, defined medium.

Cells dissociated from cerebral hemispheres of 8-day-old chick embryos were seeded on poly-L-lysine coated Petri dishes in serum-containing medium. After 24 hr the culture medium was switched to a serum-free, chemically defined medium. These cultures contain mainly neuronal cells until day 14, characterized by the presence of acetylcholinesterase activity and neurofilament proteins. After 2 weeks glial cells progressively contaminated the neuronal culture. Cultures were maintained for a period of 4 weeks. From day 6 on numerous synapses with clear vesicles were observed. The activity of choline acetyltransferase remained low throughout the culture period, while GABA levels increased in parallel with synaptogenesis. Our observations indicate that chick cerebral hemisphere neuronal cultures grown in serum-free, chemically defined medium contain GABAergic neurons that undergo maturation.

Animals↗
PubMed1987

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues.

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.

Amino Acids↗
PubMed1987

Chain-fluorinated polyamines as tumor markers--I. In vivo transformation of 2,2-difluoroputrescine into 6,6-difluorospermidine and 6,6-difluorospermine.

1. Injection of 2,2-difluoroputrescine (DFPut) into the yolk sac of chick embryos causes the formation of 6,6-difluorospermidine (6,6DFSpd) and 6,6-difluorospermine (6,6DFSpm), demonstrating that the difluoroanalogs of putrescine and spermidine are in vivo substrates of spermidine and spermine synthase, respectively. 2. Depletion of tissue putrescine and spermidine concentrations by administration of D,L-alpha-difluoromethylornithine (DFMO, Ornidyl) causes a very marked enhancement of difluoropolyamine formation from DFPut. 3. The major accumulation of 6,6DFSpd and 6,6DFSpm in DFMO-pretreated rodents occurs in small intestines and tumors, i.e. in tissues with high cell proliferation rates, which are also the most susceptible to polyamine depletion by inhibition of ornithine decarboxylase. 4. Their preferential accumulation in tumors and the fact that DFPut and its metabolites seem not to exert toxic effects, suggest DFPut as a serious candidate for the use as probe in 19F-NMR imaging of tissues with a high proliferation rate and a high rate of polyamine biosynthesis.

Animals↗
PubMed1987

Putrescine derivatives as substrates of spermidine synthase.

1. Derivatives of 1,4-butanediamine (putrescine) were studied in vitro and in vivo as potential substrates of spermidine synthase. 2. Substituents in the 1-position decreased the reaction rate by steric hindrance, and in the case of electron withdrawing groups there was an additional decrease due to the lowered basicity of the vicinal amino group. 3. Substituents in the 2-position are tolerated; under saturating conditions reaction rates are comparable to those of putrescine. 4. Compounds which were identified as substrates of spermidine synthase in vitro formed derivatives of spermidine and spermine in vivo. Exception: compounds, such as 1-methylputrescine formed in vivo only a spermidine derivative, because the second aminopropylation was sterically hindered by the substituent on the carbon atom next to the amino group. 5. Administration of 2-hydroxyputrescine to alpha-difluoromethylornithine-pretreated chick embryos produced spermidine and spermine analogues in amounts exceeding spermidine and spermine formation from putrescine under comparable conditions. 6. Since the concentration of 2-hydroxyputrescine in the embryo was higher than that of putrescine and all other putrescine analogues, it appears that uptake of the polyamine precursor from the yolk may be rate limiting. 7. Three days after administration of 5 mM alpha-difluoromethylornithine there is a near-to-complete arrest of embryonal growth. 8. A series of diamines supported growth under these conditions, even if they were not substrates of spermidine synthase. 9. Survival of chick embryos was, however, only supported if the diamines were capable of forming significant amounts of spermidine and spermine analogues.

Animals↗
PubMed1987

Functions of polyamine acetylation.

Acetylation is a means to decrease the net positive charge of the polyamines and thus liberate polyamines from anionic binding sites. The acetyl derivatives can be removed from the cells by transport and catabolism. Intracellular polyamine metabolism can be formulated as a cyclic process, which explains the transformation of one polyamine into another. As a net result, this pathway metabolizes (in an energy-requiring manner) methionine to 5'-deoxy-5'-methylthioadenosine and beta-alanine, and thus appears to be futile. It is suggested that the cyclic process is necessary for the precise control of cellular polyamine concentrations, as it allows relatively rapid spermine and spermidine concentration changes, in spite of a slow basal turnover rate. For the regulation of cellular polyamine metabolism, two decarboxylases, L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase; the cytosolic acetyl-CoA:spermidine/spermine N1-acetyltransferase; and a polyamine transport system are required. The activity of the nuclear acetyltransferase is assumed to be the rate-limiting enzyme of nuclear polyamine turnover. The complexity and high level of sophistication of polyamine regulation is strong evidence for the important functional significance of the natural polyamines.

Acetylation↗
PubMed1986

Polyamines.

Putrescine, spermidine, spermine and their derivatives are considered to be potential markers of certain diseases. Routine methods have, therefore, been developed in the course of the last decade. Several automated liquid chromatographic and gas chromatographic methods are presently at our disposal, which meet the practical requirements of sensitivity and specificity. They make the routine application of gas chromatographic-mass spectrometric methods dispensable. Very recently the amino acids deriving from polyamines have also been taken into consideration as tumour markers. Their separation by capillary gas chromatography has been reported. Separation of ion pairs on reversed-phase columns may also prove useful in the establishment of complete concentration profiles of polyamines and their derivatives in tissues and body fluids.

Animals↗
PubMed1986

Stereoselective uptake of the GABA-transaminase inhibitors gamma-vinyl GABA and gamma-acetylenic GABA into neurons and astrocytes.

The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomers R- and S-GVG and R- and S-GAG it could be shown that in both cell types only the S-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system for S-GVG (Km 78.2 +/- 20.3 microM; Vmax 0.71 +/- 0.06 nmol.min-1.mg-1 cell protein). In case of S-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes. S-GVG and S-GAG were found to be weak inhibitors of GABA uptake suggesting that S-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake of S-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T to S-GVG.

4-Aminobutyrate Transaminase↗
PubMed1986

Developmental aspects of polyamine interconversion in rat brain.

In the mammalian organism putrescine is formed by two reactions: (a) decarboxylation of ornithine and (b) degradation of spermidine via the so-called interconversion pathway. The latter comprises N1-acetylation of spermidine by a cytosolic acetyltransferase, and oxidative splitting of N1-acetylspermidine to putrescine by polyamine oxidase (PAO). It has previously been shown that specific inhibition of PAO causes a time-dependent accumulation of N1-acetylspermidine in brain, which is a measure of spermidine turnover. Another consequence of PAO inhibition is the decrease of brain putrescine concentration, proportional to its normal formation from spermidine. This observation allowed us to demonstrate the increasing significance of polyamine interconversion with brain maturation. The results support our hypothesis that the mechanisms which regulate cellular polyamine concentrations change during normal brain maturation from a system in which L-ornithine decarboxylase is dominating to a more sophisticated system in which both synthetic and catabolic processes become equally important regulatory factors. In contrast with current views, the activity of S-adenosylmethionine decarboxylase rather than that of ornithine decarboxylase limits the rate of polyamine biosynthesis during early brain development. In the mature brain the total amount of putrescine, which is formed both by decarboxylation of ornithine and by degradation of spermidine, limits the rate of spermidine formation. Changes of the regulatory system analogous to those described in this work are presumably not exclusive for brain, but rather characteristic for a variety of differentiating cells.

Aging↗