Abstracts of papers (2002)

Last Update: 08/28/2003

Abstracts of papers (2002)

[2002-1] Nishimura, K. et al., Genes Cells 7, 41-47 (2002)

Backgraound: S-adenosylmethionine decarboxylase (AdoMetDC) is one of the key enzymes involved in the biosynthesis of spermidine and spermine, which are essential for normal cell growth. To examine the role of polyamines in embryogenesis, we carried out targeted disruption of the mouse Amd1 gene, encoding AdoMetDC, to generate mice that can not synthesize spermidine and spermine.
Results: Amd1 heterozygous mice were viable, normal and fertile. However, homozygous Amd1-/- embryos died early in embryonic development, between E3.5 and E6.5 days post-coitus. Homozygous (Amd1-/-) blastoxysts at E3.5 arrested cell proliferation immediately after the onset of cell culture, and this arrest was rescued by the addition of spermidine. Chromosomal DNA breakage did not occur in Amd1-/- blastoxysts at E3.5, as determined by TUNEL assay.
Conclusions: These results indicate that AdoMetDC plays an essential role in embryonic development and that polyamines are required for cell proliferation in the embryo after E3.5.

[2002-2] Kashiwagi, K. et al., Mol. Pharmacol. 61, 533-545 (2002)

A large number of structurally diverse compounds act as open-channel blockers of NMDA receptors. They may share discrete or overlapping binding sites within the channel. In this study, the effects of mutations in and around the membrane-spanning and pore-forming regions of NMDA receptors subunits were studied with three blockers, MK-801, memantine, and TB-3-4, using recombinant NMDA receptors expressed in Xenopus laevis oocytes. Mutations at the critical asparagine residues in the M2 loop of NR1 and NR2B and at a tryptophan residue in M2 of NR2B reduced block by MK-801, memantine, and TB-3-4. Mutations at residues in the pre-M1, M1, M3, post-M3, and pot-M4 regions had differential effects on the three blockers. Many mutations in these regions reduced block by MK-801 and TB-3-4 but had no effect on block by memantine. The differential effects on block by memantine and MK-801 are unlikely to be caused by differences in the size of these blockers. Benzyl rings in MK-801 and TB-3-4 may make hydrophobic interactions with aromatic and hydrophobic amino acid residues in the pore. Some mutations in the pre-M1 and M3 regions generated constitutively open channels, characterized by large holding currents. The effects of the various mutants are discussed in the context of models based on the known structure of the pore of the KcsA potassium channel and on previous studies dealing with solvent accessible residues in NMDA receptors subunits as determined by modification after cysteine mutagenesis.

[2002-3] Nishimura, K. et al., Biochem. J. 363, 761-768 (2002)

The mechanism of inhibition of cell growth by deoxyspergualin was studied using mouse mammary carcinoma FM3A cells. Results of studies using deoxyspergualin analogues showed that both the guanidinoheptanate amide and glyoxyspermidine moieties of deoxyspergualin were necessary to cause inhibition of cell growth. When deoxyspergualin was added to the medium, there was a strong inhibition of cell growth and formation of active eukaryotic translation initiation factor 5A (eIF5A) at the third day of culture. There was also a marked decrease in cellular putrescine content and a small decrease in spermidine content. Accumulation of decapped mRNA, which is typically associated with eIF5A deficiency in yeast, was also observed. The inhibition of cell growth and the formation of active eIF5A was not reversed by addition of spermidine. The activity of deoxyhypusine synthase, the first enzyme in the formation of active eIF5A, was inhibited by deoxyspergualin in a cell-free system. These results, taken together, indicate that inhibition of active eIF5A formation is strongly involved in the inhibition of cell growth by deoxyspergualin.

[2002-4] Nitta, T. et al., Exp. Cell Res. 276, 120-128 (2002)

Polyamines, namely putrescine, spermidine, and spermine, are essential for cell survival and proliferation. A decrease in intracellular polyamine levels is associated with apoptosis. In this study, we used inhibitors of polyamine biosynthesis to examine the effect of polyamine depletion. A combination of inhibitors of ornithine decarboxylase, S-adenosylmethionine decarboxylase, or spermidine synthase decreased intracellular polyamine levels and induced cell death in a WEHI231 murine B cell line. These cells exhibited apoptotic features including chromatin condensation and oligonucleosomal DNA fragmentation. Addition of exogenous polyamines reversed the observed features of apoptotic cell death. Similar effects were also observed in other cell lines: a human B cell line Ramos and a human T cell line Jurkat. Depletion of polyamines induced activation of caspase-3 and disruption of the mitochondrial membrane potential (Dym). Inhibition of caspase activities by an inhibitor prevented the apoptotic nuclar changes but not Dym disruption induced by polyamine depletion. Overexpression of Bcl-XL, an anti-apoptotic Bcl-2 family protein, completely inhibited Dym disruption, caspase activation, and cell death. These results indicate that the depletion of intracellular polyamines tirggers the mitochondria-mediated pathway for apoptosis, resulting in caspase activation and apoptotic cell death.

[2002-5] Hirokawa, G. et al., EMBO J. 21, 2272-2281 (2002)

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post-termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.

[2002-6] Kashiwagi, K. et al., J. Biol. Chem. 277, 24212-24219 (2002)

The ATPase activity of PotA, a component of the spermidine-preferential uptake system consisting of PotA, -B, -C, and -D, was studied using purified PotA and a PotABC complex on inside-out membrane vesicles. It was found that PotA can form a dimer by disulfide cross-linking but that each PotA molecule functions independently. When PotA was associated with the membrane proteins PotB and PotC, the Km value for ATP increased and PotA became much more sensitive to inhibition by spermidine. It was also shown that spermidine uptake in cells was gradually inhibited in parallel with spermidine accumulation in cells. The results suggest that spermidine functions as a feedback inhibitor of spermidine transport. The function of PotA was analyzed using PotA mutants obtained by random mutagenesis. There are two domains in PotA. The NH2-terminal domain (residues 1-250) contains the ATP binding pocket formed in part by residues Cys26, Phe27, Phe45, Cys54, Leu60, and Leu76, the active center of ATPase that includes Val135 and Asp172, and amino acid residues necessary for the interaction with a second PotA subunit (Cys26) and with PotB (Cys54). The COOH-terminal domain (residues 251-378) of PotA contains a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity.

[2002-7] Kawano, M. et al., J. Biol. Chem. 277, 24405-24410 (2002)

The 76-kDa NtpI subunit constitutes the membrane-embedded V0 moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337-48340). The negative cooperativity for the Na+ dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 ア 0.03 and 0.40 ア 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentraitons approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and or the cooperative features of E. hirae vacuolar-type ATPase.

[2002-8] Kawano, M. et al., Biosci. Biotechnol. Biochem. 66, 1597-1600 (2002)

We here isolated an Enterococcus hirae mutant unable to grow well at pH 10. The influx rate calculated from steady-state 42K+/K+ exchange and the intracellular K+ concentration of the mutant were reduced to 53 and 55% of those of the wild-tpe, respectively. The activities of two high-affinity K+ uptake systems, KtrI and KtrII, were normal in the mutant, but the kinetics of net K+ uptake at pH 10 indicated that a low-affinity K+ uptake with a Km of about 2 mM (Kawano, M, Abuki, R, Igarashi, K, Kakinuma, Y. (2001) Arch. Microbiol. 175: 41-45), which were seen in the wild-type, was deficient in this mutant.

[2002-9] Yasumura, K. et al., Arch. Microbiol. 178, 172-179 (2002)

The Enterococcus hirae ntp operon encodes all subunits of the vacuolar-type ATPase (V-ATPase), which transports Na+ or Li+. This operon is expressed preferentially in response to Na+, but not to Li+. Deletion analysis of the ntp promoter region in plasmids indicated that the AT-tract between -198 and -132 is required for Na+-specific transcriptional regulation. In addition, lithium-tolerant (LTR) mutants were isolated in which functional V-ATPase levels were high even in Na+-depleted medium. Western blot and Northern blot experiments revealed an increase in basal Na+-independent transcription in one of the mutants (LTR1). The nucleotide sequences of the ntp promoter region of the LTR mutants showed mutational conversion of single base-pairs between positions -23 and +1. Na+-independent expression of a reporter gene linked to the ntp promoter in plasmids was elevated by base substitutions at -23 to +1, and promoter activity induced by these base substitutions was lost by deletion of the region between -198 and -132. These results suggest that the AT-tract between -198 and -132 is indispensable for transcription of the ntp operon.

[2002-10] Hirokawa, G. et al., J. Biol. Chem. 277, 35847-35852 (2002)

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg2+ and NH4+ ions in a similar manner.

[2002-11] Yoshida, M. et al., J. Biol. Chem. 277, 37139-37146 (2002)

The mechanisms by which polyamines stimulate synthesis of the RNA polymerase sigma38 subunit in Escherichia coli were studied. Polyamine stimulation was observed only in strains in which the 33rd codon of RpoS mRNA is a UAG termination codon instead of a CAG codon for glutamine in wild-type E. coli. Readthrough of the termination codon by Gln-tRNAsupE was stimulated by polyamines. This stimulation was found to be caused by an increase in both the level of suppressor tRNAsupE and the binding affinity of Gln-tRNAsupE for ribosomes. The stimulatory effect was observed with a UAG termination codon but not with UGA and UAA codons. Readthrough of the UAG termination codon at the 270th amino acid position of RpoS mRNA was also stimulated by polyamines, indicating that polyamines stimulate readthrough of a UAG codon regardless of its location within the RpoS mRNA. When cell viability of an E. coli strain having a termination codon in the 33rd position of RpoS mRNA was compared using cells cultured with or without putrescine, it was higher in cells cultured with putrescine than in cells cultured without putrescine. The level of sigma38 subunit in the cells cultured with putrescine was higher than that in cells cultured without putrescine on days 2, 4, and 8, but the level of sigma70 subunit was almost the same in cells cultured with or without putrescine. These results confirm that elevated expression of the rpoS gene is important for cell viability at late stationary phase.

[2002-12] Raj, V. S. et al., Biochem. Biophys. Res. Commun. 299, 252-257 (2002)

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma38 subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma38), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.

[2002-13] Samata, K. et al., Cancer Chemother. Pharmacol. 50, 367-372 (2002)

We established an NC-190-resistant cell line, FM/NC-R, from the murine mammary carcinoma cell line FM3A and examined some of its characteristics. FM/NC-R cells were prepared by mutagen treatment followed by exposure to NC-190 in the culture medium. FM/NC-R cells were 76.5 times more resistant against NC-190 than FM3A cells as measured by their growth in vitro. FM/NC-R cells also showed cross-resistance to etoposide with NC-190. Neither NC-190 nor etoposide increased the lifespan of FM/NC-R-bearing mice at doses that prolonged the lifespan of FM3A-bearing mice more than four times. This resistance was not due to the change in the concentration of NC-190 in the cells, and there was no change in the expression of P-glycoprotein, a drug efflux pump in the cells. NC-190 and etoposide are inhibitors of DNA topoisomerase II, but there was no difference in cellular content of DNA topoisomerase II between the two cell lines as determined by Western blot analysis. The stabilization of DNA-DNA topoisomerase II cleavable complexes induced by NC-190 was lost in FM/NC-R cells. It was found that Gly881, which is located in the ATP binding site, was replaced by Arg in topoisomerase IIalpha of FM/NC-R cells. These results indicate that the NC-190-resistant cell line FM/NC-R contains a mutated DNA topoisomerase IIalpha.

[2002-14] Tomitori, H. et al., Biochim. Biophys. Acta 1579, 180-184 (2002)

Spermidine/spermine N1-acetyltransferase (SSAT), the key enzyme of polyamine catabolism, is induced by antiproliferative stresses. We analyzed the 5' flanking region of the human SSAT gene, and clarified that the binding of Sp1 to the GC-box located 42 to 51 bp upstream responsible for the elevated transcription after X-ray irradiation.

[2002-15] Sumino, M. et al., Chem. Pharm. Bull. 50, 1484-1487 (2002)

Novel alkylphenols, ardisiphenols A-C (1-3) and a novel bergenin derivative, demethoxybergenin (10) were isolated from the fruits of Ardisia colorata (Myrsinaceae), together with known alkylresorcinols (4-6), embelin (7), myricetin (8), quercetin (9), bergenin (11), norbergenin (12), kaempferol (13), quercetin-3-O-beta-D-glucopyranoside (14) and gallic acid (15). Their structures were determined by NMR, MS(/MS) analyses and other spectroscopic methods. Ardisiphenols showed moderate scavenging activities toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and showed cytotoxicity against the murine breast cancer cell line, FM3A.

[2002-16] Gao, F. et al., Life Sci. 72, 669-676 (2002)

A synthetic analog of prostaglandin E1, OP-1206 [17S, 20-dimethyl-trans-delta2-prostaglandin E1] protects the small intestine from the methotrexate (MTX)-induced damage. The purpose of this study is to evaluate the protective effect of OP-1206 on the methotrexate-induced small intestinal damage in rats from the biochemical point of view. MTS (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days, and on the 6th day biochemical components in the jejunal mucosa of the treated rats were determined. The contents of DNA, RNA, proteins and polyamine (spermine and spermidine) in the jejunal mucosa of rats were markedly decreased by the MTX treatment. The coadministration of OP-1206 with MTX prevented it. These results indicated that OP-1206 could protect the intestinal mucosa against the biochemical effects of MTX through a trophic action on intestinal villi. Further, it should be noted that polyamines may possibly play an important role of modulation action on intestinal mucosa.

[2002-17] Samata K. et al., Res. Commun. Mol. Path. Pharmacol. 111, 77-87 (2002)

The novel antitumor compound NC-190 strongly inhibited the growth of FM3A cells with an IC50 of 0.019 [micro]g/ml (0.042 [micro]M) when cultured with NC-190 for 48 h. NC-190 potently suppressed DNA synthesis with 90% inhibition observed at 0.1 [micro]g/ml of NC-190. RNA and protein syntheses were also suppressed under the same conditions, but to a lesser extent. We then measured the cellular enzymatic activities of DNA polymerase alpha, RNA polymerase, thymidine kinase, thymidylate synthase and Leu-tRNA synthetase of FM3A cells cultured with or without NC-190. Of these 5 enzymes, the activity of thymidine kinase was most strongly suppressed by NC-190, by 77%. Although NC-190 did not directly inhibit the activity of thymidine kinase in a cell-free system, expression of mRNA of thymidine kinase was suppressed by 75% in NC-190-treated cells. These results indicate that NC-190 can suppress the expression of the gene for thymidine kinase and the inhibition of thymidine kinase contributes to the inhibition of cell growth by NC-190 together with the inhibition of topoisomerase II.