Abstracts of papers (2008)

Last Update: 11/26/2008

Abstracts of papers (2008)

[2008-1] Higashi, K. et al., J. Bacteriol. 190, 872-878 (2008)

A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr(4), Trp(5), Glu(15), Tyr(45), Tyr(61), and Glu(82) in MdtJ and Glu(5), Glu(19), Asp(60), Trp(68), and Trp(81) in MdtI are involved in the excretion activity of MdtJI.

[2008-2] Hamamoto, S. et al., J. Biol. Chem. 283, 1911-1920 (2008)

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 [micro]M Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.

[2008-3] Higashi, K. et al., Biochem. Biophys. Res. Commun. 370, 572-577 (2008)

Polyamines are essential for cell growth due to effects mainly at the level of translation. These effects likely involve a structural change, induced by polyamines, of the bulged-out region of double-stranded RNA that is different from changes induced by Mg(2+). Structural changes were studied using U6-34, a model RNA of U6 small nuclear RNA containing bulged nucleotides. Binding of NS1-2 peptide derived from the RNA binding site of NS1 protein, to U6-34 was inhibited by spermidine but not by Mg(2+). A selective conformational change of the bases in the bulged-out region of U6-34 induced by spermidine was observed by NMR. The selective effect of spermidine was lost when the bulged-out region of U6-34 was removed in U6-34(Delta5). The binding of NS1-2 peptide to U6-34(Delta5) was inhibited both by spermidine and Mg(2+). The selective structural change of U6-34 by spermidine was confirmed by circular dichroism.

[2008-4] Shiokawa, K. et al., Gene Regul. Syst. Biol. 2, 1-19 (2008)

S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethionine (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage of SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation by caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastcoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extent of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a "fail-safe" mechanism of early development to save the embryo from accidental damages that take place during cleavage.

[2008-5] Ugai, K. et al., J. Toxicol. Sci. 33, 237-240 (2008)

Cytosine and adenine (CA) repeats polymorphism (D14S1026) iterating "cytosine and adenine" nucleotide motifs is one of the genomic microsatellites in intron 5 of the estrogen receptor beta (ER beta) gene (14q22-24). Relations between CA repeats polymorphism and several diseases have been shown. Although the relation between number of CA repeats and gene transcription has been actively studied using several genes, results have remained contradictory until this time. In this study, we examined the functional effects of CA repeats polymorphism on transcriptional activity based on our knowledge of the ER beta gene. After preparing four types of reporter gene constructs containing 15, 18, 24 or 27 CA repeats, luciferase reporter gene assays were performed. Relative luciferase activities of these constructs were not significantly different from that of the no inserted vector and variation of CA repeats did not affect these activities. Our results indicate that CA repeats polymorphism might not only affect transcriptional activity but also other processes of gene expressions. Further studies are needed to clarify the specific functions of CA repeats polymorphism in the ER beta gene.

[2008-6] Masuko, T. et al., Neurochem. Sci. 53, 38-44 (2008)

The linear polyamine spermine enhances N-methyl-d-aspartate (NMDA) receptors activity at depolarized membrane potential and shows a voltage-dependent block. Spermine potentiates NMDA receptor currents in the presence of saturating concentrations of glutamate and glycine, but cyclic polyamines such as CP2323 do not. CP2323 inhibited the currents most potently amongst 10 kinds of cyclic polyamines tested. The inhibition was prominent at heteromeric NR1/NR2A and NR1/NR2B receptors but not at NR1/NR2C and NR1/NR2D receptors expressed in Xenopus oocytes. Inhibition by CP2323 was voltage-dependent, because the degree of inhibition was in the order -100 mV > -70 mV > -20 mV. It was 10-100 times more prominent than inhibition by spermine. The inhibitory potency of both CP2323 and spermine was attenuated by the mutations around the vestibule of the channel pore at NR1 W563, N650, T807, and NR2B Y646. Inhibition by CP2323 was hardly affected by the mutations of NR1 N616 and E621, whereas inhibition by spermine was reduced by these mutations. The results suggest that CP2323 interacts with the vestibule region of the NMDA receptor and does not enter deep into the channel. Mutations of NR2B W607 greatly reduced the inhibition by CP2323 and spermine, suggesting that the mutation of this residue may cause the change of the channel structure. Neuroprotective effects of cyclic polyamines against cell damage caused by NMDA were compared with those of spermine in cultured rat hippocampal neurons. Addition of CP2323, but not spermine, into the medium attenuated the neurotoxicity induced by NMDA. These results indicate that CP2323 functions as a channel blocker of the NMDA receptor.

[2008-7] Jin, L. et al., J. Pharmacol. Exp. Ther. 327, 68-77 (2008)

The transmembrane and pore-forming regions of N-methyl-D-aspartate receptors containing the NR1 and NR2B subunits were studied by measuring the effects of various NR1 and NR2B mutants on stimulation and block by spermine. Block by spermine was predominantly affected by mutations in the M3 segment of NR1 and especially in the M1 and M3 segments of NR2B. These regions are in the outer vestibule of the channel pore and may contribute to a spermine binding site. Mutations in different regions-predominantly the M3 segment and M2 loop of NR1 and the M3 segment of NR2B-influenced spermine stimulation, a surprising finding because spermine stimulation is thought to involve a spermine binding site in the distal, extracellular regulatory domain. However, some of these mutations also influence sensitivity to ifenprodil and protons, and changes in spermine sensitivity may be secondary to changes in proton sensitivity. The results are consistent with the proposal that the relative positions of the M1 and M3 transmembrane segments and M2 loops are staggered or asymmetric in NR1 and NR2 subunits, and with the idea that stimulation and block by spermine involve separate binding sites and distinct mechanisms, although some residues in the receptor subunits can affect both stimulation and block.

[2008-8] Higashi, K. et al., J. Biol. Chem. 283, 32989-32994 (2008)

Polyamines play important roles in cell growth mainly through their interaction with RNA. We have previously reported that polyamines stimulate the synthesis of oligopeptide binding protein (OppA) in Escherichia coli and the formation of Ile-tRNA in rat liver. These effects involve an interaction of polyamines with the bulged-out region of double-stranded RNA in the initiation region of OppA mRNA and in the acceptor stem of rat liver tRNA(Ile). In the present study, effects of polyamines on E. coli OppA synthesis and rat liver Ile-tRNA formation were compared by using OppA mRNA and tRNA(Ile) with or without the bulged-out region of double-stranded RNA. The results indicate that the bulged-out region is involved in polyamine stimulation of OppA synthesis and Ile-tRNA formation. A selective structural change by spermidine of the bulged-out region of double-stranded RNA was confirmed by circular dichroism.

[2008-9] Han, X. et al., J. Neurochem. 107, 1566-1577 (2008)

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-D-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were over-expressed in Escherichia coli and purified to near homogeneity. The Kd values for binding of [(14)C]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 [micro]M, respectively. [(3)H]Ifenprodil bound to NR1-R (Kd, 0.18 [micro]M) and NR2B-R (Kd, 0.21 [micro]M), but not to NR2A-R at the concentrations tested (0.1-0.8 [micro]M). These Kd values were confirmed by circular dichroism measurements. The Kd values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.