PubMed Health⌕ Search

Biomedical subjects

K Samejima

Publications and source records attributed to K Samejima.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ionspray ionization-mass spectrometric separation and determination of heptafluorobutyryl derivatives of polyamines.

We describe here an application of ionspray ionization-mass spectrometry (IS-MS) for simultaneous determination of putrescine, spermidine, and spermine without the need for coupling with other separation techniques such as high-performance liquid chromatography or capillary electrophoresis. Because it was essential to increase the mass of these compounds to eliminate contaminated low-mass ions, heptafluorobutyryl (HFB) derivatives were chosen. It was found that each HFB-polyamine appeared as a single major cation carrying an ammonium ion in a solution of 50% acetonitrile-water containing 0.5% ammonium acetate at an orifice potential of 20 V. Under these conditions, the three polyamines after addition of 15N-labeled polyamines as internal standards, followed by heptafluorobutylation, were determined simultaneously. It was shown, using a sample injection bulb set between the syringe drive and ionspray probe, that the analysis time for data acquisition was about 3 min and the sensitivity was about 10 fmol. In addition, polyamines in rat liver were determined by this method after simple fractionation with a small column of CM-cellulose with reasonable accuracy and precision. Behaviors of acetylated polyamines were also described, and the results showed that the acetylated polyamines would become additional target compounds in the present method.

Animals↗
PubMed1998

Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution.

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.

Animals↗
PubMed1998

ICAD/DFF regulator of apoptotic nuclease is nuclear.

In nonapoptotic cells, the caspase-activated DNase CAD/CPAN is associated with a regulatory subunit, ICAD/DFF, that binds to it and blocks its enzymatic activity. It has been proposed that a major function of ICAD is to restrain CAD in the cytoplasm in healthy cells. The experiments presented here demonstrate that rather than being cytoplasmic, a GFP-ICAD fusion protein is nuclear in healthy human, pig, and chicken cells. Furthermore, immunoblots using antibodies to murine ICAD confirm the presence of endogenous ICAD and the marker protein DNA topoisomerase I in human nuclei, while tubulin was found solely in the cytosolic fraction. Since ICAD is located in cell nuclei, it is unlikely that the protein functions primarily in the cytoplasm either as an anchor for CAD/CPAN or as a factor that enters the nucleus following caspase cleavage in order to activate resident endonucleases.

Animals↗
PubMed1997

Structural requirement for axonal regeneration-promoting effect of polyamines in cultured rat hippocampal neurons.

We have previously found that spermine, spermidine and putrescine promote axonal regeneration following axotomy in cultured rat hippocampal neurons. In the present study, we investigated which part of the polyamine molecule is responsible for the regeneration-promoting effect. Testing the effects of several synthetic analogues revealed that the butanediamine moiety is essential for the activity and the terminal primary amines are necessary for full agonist activity. The structure-activity relationship indicates that the regeneration-promoting effects of polyamines are not associated with NMDA receptors.

Animals↗
PubMed1997

[Methodological approach to regulation of polyamine].

This brief article is dedicated to the late Professor Morizo Ishidate, and concerned in a methodology developed by the author and collaborators, aiming at the regulation of polyamine, especially, the regulation of spermidine synthase. The content is separated in 3 sections. The first section on the development of analytical methods, contains seven items, e.g. fluorometric determination of polyamines by fluorescamine, analysis of naturally occurring polyamines by GC and GC-MS, etc. The second section on the syntheses of needed compounds, contains three items, e.g. syntheses of decarboxylated S-adenosylmethionine which is a substrate for spermidine synthase and its related compounds, syntheses of 15N-enriched polyamines applicable to the preparation of various polyamines, etc. The last section on the studies of aminopropyl transferases, contains three items, e.g. purification of spermidine synthase from mammalian tissues using ATPA-Sepharose, a novel affinity carrier, newly developed inhibitors for the enzymes, etc.

Animals↗
PubMed1996

Determination of covalently bound hypusine and deoxyhypusine to protein using submilligram of protein samples by HPLC.

A sensitive and reliable method for the determination of hypusine and deoxyhypusine in eIF-5A protein, an initiation factor of protein synthesis, was developed. An advantage of this method is the use of N epsilon-(5-aminopentyl)lysine, an analogue of deoxyhypusine, as an internal standard. The application made it possible to determine hypusine in less than a mg of protein samples from cultured HTC cells and rat organs. After acid hydrolysis of protein samples to which had been added the internal standard, the hydrolysates were fractionated by carboxymethyl cellulose column chromatography. Also, diamine fractions containing a few pmol of hypusine and deoxyhypusine were successfully analyzed by a reversed phase HPLC with a fluorescence detection of o-phthalaldehyde. The method was applied for the determination of hypusine and deoxyhypusine in drug-treated HTC cells and normal rat organs. The results from HTC cells were discussed based on the known effects of each drug on hypusine biosynthesis.

Animals↗
PubMed1995

Search for novel ligands selective at a polyamine recognition domain on the N-methyl-D-aspartate receptor complex using membrane binding techniques.

Among over 60 polyamine derivatives tested, only N-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in "non-washed" rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [3H]MK-801 binding before equilibrium in the presence of L-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in "Triton-treated" membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [3H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [3H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.

Animals↗
PubMed1995

Specific depletion of spermidine and spermine in HTC cells treated with inhibitors of aminopropyltransferases.

The effects of a potent spermidine synthase inhibitor, trans-4-methylcyclohexylamine (4MCHA), and a spermine synthase inhibitor, N-(3-aminopropyl)cyclohexylamine (APCHA), on polyamine biosynthesis and cell growth have been studied in rat hepatoma cells (HTC cells) in culture. Treatment of HTC cells with 4MCHA or APCHA caused a marked decrease of spermidine or spermine with a compensatory increase of putrescine and spermine or spermidine, respectively, in a dose-dependent manner, suggesting specific and potent inhibition of each target enzyme. When 250 microM 4MCHA or APCHA was administered to the cells for 8 days, spermidine was decreased to 2% of control culture or spermine below 1%, respectively, while total polyamine (sum of putrescine, spermidine, and spermine) remained almost unchanged during the culture. There were no significant changes in the growth rate during treatment with the inhibitors at 250 microM concentration. The results suggest that in the growth of HTC cells, putrescine and spermine can be substituted for most of the fraction of cellular spermidine, and spermidine for most of the fraction of cellular spermine. Of five enzymatic activities involved in polyamine biosynthesis and interconversion, S-adenosylmethionine decarboxylase activity increased 8-fold with 250 microM 4MCHA, and 3-fold with 250 microM APCHA during the treatment. This increase was partially due to the increase of half-life of the enzyme. Separate roles for spermidine and spermine in the biosynthesis of the enzyme protein were also suggested.

Acetyltransferases↗
PubMed1995

Enzymatic aminopropylation of certain secondary amines.

Two unusual aminopropyl acceptors found in a survey of putrescine binding sites of mammalian spermidine synthase, N-methylputrescine (I) and 4-aminomethylpiperidine (II), were examined for their aminopropyl derivatives. Studies under in vitro incubation conditions suggested that the aminopropyl derivatives of the secondary amine of I and II, N4-methylspermidine (Is) and 1-N-(3-aminopropyl)-4-aminomethylpiperidine (IIs), and of the primary amine of I and II, N8-methylspermidine (Ip) and 4-[N-(3-aminopropyl)aminomethyl]piperidine (IIp), respectively, were biosynthesized by rat spermidine synthase. Studies on the cell culture system of cultured rat hepatoma (HTC) cells treated with alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor, clearly showed the presence of Is and Ip when I was administered, and IIs and IIp when II was administered, with no detection of putrescine or spermidine. These results suggested that mammalian spermidine synthase can transfer the aminopropyl moiety of decarboxylated S-adenosylmethionine to certain secondary amines in living cells.

Animals↗
PubMed1995

Structural requirement for neurotrophic activity of spermine in cultured rat hippocampal neurons.

We investigated the structure-activity relationship for the neurotrophic activity of spermine by comparing the effects of several synthetic spermine analogues on the survival of cultured rat hippocampal neurons. N,N'-bis(3-aminopropyl)-1,6-hexanediamine and N,N'-bis(3-aminopropyl)-1,3-propanediamine did not promote the neuronal survival, suggesting that the central butanediamine structure is essential for the neurotrophic activity. Furthermore, N1,N12-bis(ethyl)spermine significantly promoted the neuron survival, but its maximum effect was smaller than that of spermine, indicating that two terminal primary amines are required for full agonist activity. The structural requirement for the neurotrophic activity of spermine was different from those for any other reported activities of polyamines.

Animals↗
PubMed1995

Antagonistic effect of N-(3-Aminopropyl)cyclohexylamine on neurotrophic action of spermine in primary cultured rat hippocampal and cerebellar neurons.

We previously found that spermine potently promotes the neuronal survival and regeneration of primary cultured brain neurons. N-(3-Aminopropyl)cyclohexylamine (APCHA) was originally developed as a spermine synthase inhibitor. To test if endogenous spermine biosynthesis contributes to neuronal survival and morphogenesis, we examined the effects of APCHA in primary cultured rat hippocampal and cerebellar neurons. APCHA at concentrations up to 10(-6) M did not affect the neuronal survival, but significantly blocked the survival-promoting effect of spermine (10(-8) M). APCHA also blocked the spermine-induced promotion of neurite regeneration following axotomy. Unlike APCHA, another cyclohexylamine derivative trans-4-methylcyclohexylamine did not affect the neurotrophic effect of spermine. These results suggest that in primary cultured brain neurons, APCHA works as a spermine antagonist rather than as a spermine synthesis inhibitor.

Animals↗
PubMed1994

N-(3-aminopropyl)-cyclohexylamine blocks facilitation by spermidine of N-methyl-DL-aspartate-induced seizure in mice in vivo.

The facilitating or antagonizing effects of polyamine analogues on N-methyl-DL-aspartate (NMDLA)-induced seizures were investigated using mice. Intracerebroventricular injection of spermidine and spermine, but not putrescine, shortened the latency to appearance of clonic convulsion induced by subcutaneous administration of NMDLA. Injection of N-(3-aminopropyl)cyclohexylamine (APCHA) alone did not affect the NMDLA-induced seizure. However, APCHA, when administered together with spermidine, clearly antagonized the facilitating effect of spermidine on the NMDLA-induced seizure. Another cyclohexylamine derivative, trans-4-methylcyclohexylamine, did not block the effect of spermidine. APCHA also antagonized the facilitation by D-serine of NMDLA-induced seizure, although the blocking effect for D-serine was weaker than that for spermidine. APCHA should be useful as a new tool for pharmacological studies on the neuromodulatory action of polyamines.

Animals↗
PubMed1994

Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis.

N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities. 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA. When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced. From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA. These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding). The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2. The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA. A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities.

Antineoplastic Agents↗
PubMed1994

Occurrence of tertiary and quaternary branched polyamines in thermophilic archaebacteria.

Polyamines of thermophilic archaebacteria were analysed by high-performance liquid chromatography and gas chromatography. Thermoplasma acidophilum and Thermoplasma volcanium ubiquitously contained spermidine and spermine. Four species of Sulfolobus, S. acidocaldarius, S. solfataricus, S. metallicus and S. shibatae, two species of Acidianus, A. brierleyi and A. infernus, and Metallosphaera sedula, contained norspermidine and norspermine in addition to spermidine and spermine, but quantitative distribution profiles were species-specific. A tertiary tetra-amine, N4-aminopropylspermidine, and a quaternary penta-amine, N4-bis(aminopropyl)spermidine, were detected as a major polyamine in three species of Thermococcus, T. celer, T. litoralis and T. stetteri, and two Pyrococcus species, P. furiosus and P. woesei. This is the first report on the occurrence of branched polyamines in archaebacteria.

Archaea↗
PubMed1993

Interaction of a polyamine analogue, 1,19-bis-(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), with DNA and effect on growth, survival, and polyamine levels in seven human brain tumor cell lines.

Computer graphics modeling and physicochemical studies of spermine-DNA interactions, as well as experiments in cell culture, indicate that a polyamine analogue with strong affinity for nucleic acids but poor ability to condense and aggregate DNA in vitro should act as an antiproliferative agent if it can enter cells. On the basis of our studies of polyamine-DNA interactions, we designed a pentamine, 1,19-bis(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), that had these characteristics. Measurement of melting temperature and ultraviolet light scattering studies show that the affinity of this analogue for calf-thymus DNA is about 4 times higher than that of spermine, whereas its ability to aggregate DNA is slightly poorer than that of spermine. Studies in U-87 MG, U-251 MG, SF-126, SF-188, SF-763, SF-767, and DAOY human brain tumor cells in tissue culture showed that treatment for more than 96 h with concentrations of 5 microM BE-4-4-4-4 or greater inhibited growth; decreased levels of putrescine, spermidine, and spermine; and decreased colony-forming ability in all cell lines. The cytotoxicity of the analogue varied among cell lines; DAOY and SF-767 were the most sensitive and the most resistant lines, respectively. In SF-763 cells, growth inhibition by BE-4-4-4-4 could be partially reversed by the addition of putrescine, spermidine, or spermine 1 day after BE-4-4-4-4 addition, but in U-251 MG cells, growth inhibition was reversed only by spermine and not by other polyamines. When any of the naturally occurring polyamines was added simultaneously with BE-4-4-4-4, growth inhibition was completely blocked. The data suggest that a threshold intracellular concentration of BE-4-4-4-4 is needed to manifest the growth-inhibitory and cytotoxic effects. In most cell lines, once that threshold level is reached, the growth-inhibitory and cytotoxic properties of the analogue are manifest irrespective of cellular polyamine levels. Further increases in the BE-4-4-4-4 concentration or incubation time reduce the intracellular polyamine levels but do not significantly increase growth inhibition. In U-87 MG and DAOY cells, however, prolonged incubation with higher concentrations of BE-4-4-4-4 causes additional growth inhibition along with depletion of intracellular polyamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain Neoplasms↗
PubMed1993

Effects of inhibitors of spermidine synthase and spermine synthase on polyamine synthesis in rat tissues.

Several inhibitors of aminopropyltransferases, developed recently in this laboratory, were tested for their specificity by measuring their effects on six enzyme activities related to polyamine biosynthesis and interconversion. Two of them, trans-4-methylcyclohexylamine (4MCHA) and N-(3-aminopropyl)cyclohexylamine (APCHA), selectively and potently inhibited the activities of spermidine synthase and spermine synthase, respectively. They were subjected to in vivo studies using rats. Oral administration of 4MCHA or APCHA dissolved in drinking water (0.02 and 0.1%) available ad lib. for a period of 10 days or 4 months caused a specific and marked decrease in spermidine or spermine in tissues (such as a 95% decrease) with a compensatory increase of spermine or spermidine, respectively, but without any observable change in the growth of the treated rats. Also, with extreme reduction of spermidine or spermine, when their sum was approximately constant, the activity of S-adenosyl-methionine decarboxylase in these tissues was enhanced significantly with no change in the activity of ornithine decarboxylase. These results suggested a separate role for spermidine or spermine in the in vivo enhancement of S-adenosylmethionine decarboxylase activity.

Adenosylmethionine Decarboxylase↗
PubMed1993

Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test.

Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec-) double mutant mei-9a mei-41D5 males and Rec+ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level; the experiment was repeated with a larval stock consisting of Rec+ males and Rec+ females under comparable conditions. A preferential killing effect upon Rec- larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons.

2-Acetylaminofluorene↗