Abstracts of papers (2004)

Last Update: 01/11/2005

Abstracts of papers (2004)

[2004-1] Yasumura, K. et al., Br. J. Haematol. 124, 179-185 (2004)

Ikaros is a Kruppel-type zinc finger protein that is essential for normal lymphocyte development and differentiation. Recently, it has been demonstrated that Ikaros is frequently inactivated in both human and mouse leukaemias/lymphomas. Although this inactivation is thought to be involved in leukaemogenesis, little is known about the molecular mechanisms that lead to neoplastic transformation. To identify the genes that may be controlled by Ikaros, we performed differential display analysis of RNAs from mouse 3T3-L1 cells that had been transfected with the Ikaros gene. Two cDNAs, the Trk-fused gene (Tfg) and death-associated protein 3 gene (Dap3) were upregulated in Ikaros-transfected cells. Expression of Tfg and Dap3 was consistently downregulated in radiation-induced T-cell lymphomas that exhibited defective Ikaros expression. These results suggest that Tfg and Dap3 function downstream of Ikaros and may be involved in radiation-indeuced lymphomagenesis.

The effects of low concentrations of methotrexate (MTX) on the growth of suspension (FM3A, 2B4 and THP-1) and adherent (NIH3T3 and V79) cells were compared. The concentration of methotrexate to cause the inhibition of cell growth was lower in suspension cells than in adherent cells. The IC(50) for FM3A, 2B4, THP-1, NIH3T3 and V79 cells were 3.5, 5, 9, 30 and 50 nM, respectively. The inhibition of cell growth was reversed completely by tetrahydrofolate and was fully or significantly reversed by adenosine and thymidine, suggesting that the effects of low concentrations of methotrexate result from the inhibition of biosynthesis of purines and pyrimidines. In suspension cells but not in adherent cells there was a decrease in the levels of S-adenosylmethionine and polyamines after methotrexate treatment. Growth of suspension but not adherent cells was significantly recovered by treatment with S-adenosylmethionine. However, treatment with spermidine did not reverse the effects of methotrexate in any of the cell lines. The preferential inhibitory effect of methotrexate in suspension cells versus adherent cells was due mainly to a more rapid uptake of methotrexate. This may be relevant to the in vivo effects of low doses of methotrexate, which have immunosuppressive and anti-inflammatory effects, because lymphocytes are suspension cells .

[2004-3] Soksawatmaekhin, W. et al., Mol. Microbiol. 51, 1401-1412 (2004)

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.

[2004-4] Uemura, T. et al., Biochem. Biophys. Res. Commun. 315, 1082-1087 (2004)

The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12 h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.

[2004-5] Kashiwagi, K. et al., J. Pharmacol. Exp. Ther. 309, 884-893 (2004)

The effects of various anthraquinone polyamines (AQP) were studied at recombinant N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus laevis oocytes. The AQP derivatives had different numbers of methylene groups between the NH(2) (or NH) groups in their spermidine-like tail. Thus, we termed these derivatives AQ33, AQ34, etc. All AQP derivatives inhibited responses of NR1/NR2 receptors in oocytes voltageclamped at -70 mV, with IC(50) values between 4 and 22 microM. The block was strongly voltage-dependent. AQ34 and AQ33b inhibited responses of NR1/NR2 receptors but did not inhibit responses of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors expressed from GluR1 or GluR2(Q), indicating that AQ34 and AQ33b are preferential NMDA antagonists. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by AQ34 and AQ33b. These residues are located in the outer vestibule at the selectivity filter/narrowest constriction of the channel and in the inner vestibule below the level of the selectivity filter. The results with mutant NR1 and NR2 subunits are consistent with the idea that NR1(Asn616) and NR2B(Asn616), but not NR2B(Asn615), make the narrowest constriction of NMDA channel.

[2004-6] Kusama-Eguchi, K. et al., Biol. Pharm. Bull. 27, 1052-1058 (2004)

Neurolathyrism is a human motoneuron disease caused by the overconsumption of grass pea (Lathyrus sativus) that contains a toxic non-protein amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-beta-ODAP). The preventive activities of various glutamatergic agents from acute neuronal death caused by L-beta-ODAP were studied using rat primary cortical neuron/glia culture. Nearly 80% of the rat primary cortical neurons were killed by 300 [micro]M L-beta-ODAP within 24 h. Though antagonists acting on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor prevented most of the toxicity, antagonists acting on group I metabotropic glutamatergic receptors (mGluRs), including (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), (S)-alpha-methyl-4-carboxyphnylglycine (MCPG), and 2-methyl-6-(2-phenylethenyl)pyridine (SIB1893) partially and significantly prevented neuronal death due to L-beta-ODAP. These antagonists, within limited concentrations, did not have any inhibitory effects on the currents through AMPA receptors expressed in Xenopus oocytes. These results suggest that the neurotoxicity induced by L-beta-ODAP is partially mediated by the activation of groupI mGluRs by an indirect mechanisms.

[2004-7] Fukuchi, J. et al., J. Biol. Chem. 279, 29921-29929 (2004)

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

[2004-8] Yoshida, M. et al., J. Biol. Chem. 279, 46008-46013 (2004)

We reported previously that the synthesis of specific proteins such as OppA, Cya, and RpoS ( sigma(38) ), which are important for cell growth and viability, is stimulated by polyamines at the level of translation. In this study we found that the synthesis of FecI and Fis was also stimulated by polyamines at the level of translation. The FecI and Fis proteins enhance the expression of mRNAs that are involved in iron uptake and energy metabolism and the expression of rRNA and some tRNAs. The Shine-Dalgarno (SD) sequence of their mRNAs was not obvious or was not located at the usual position. When the SD sequences were created at the normal position on these mRNAs, protein synthesis was no longer influenced by polyamines. Thus, the common characteristic of these mRNAs was to have a weak or ineffective SD sequence. We propose that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as a "polyamine modulon." By DNA microarray, we found that 309 of 2,742 mRNA species were upregulated by polyamines. Among the 309 up-regulated genes, transcriptional enhancement of at least 58 genes might be attributable to increased levels of the transcription factors Cya, RpoS, FecI, and Fis, which are all organized in the polyamine modulon. This unifying molecular mechanism is proposed to underlie the physiological role of polyamines in controlling the growth of Escherichia coli .

[2004-9] Nakatsu, F. et al., J. Cell Biol. 167, 293-302 (2004)

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B(-/-) mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B(-/-) mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

[2004-10] Hirokawa, G. et al., Mol. Microbiol. 54, 1011-1021 (2004)

The post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Without RRF, the ribosome is not released from mRNA at the termination codon and reinitiates translation downstream. This is called unscheduled translation. Here, we show that at the non-permissive temperature of a temperature-sensitive RRF strain, RRF is lost quickly, and some ribosomes reach the 3' end of mRNA. However, instead of accumulating at the 3' end of mRNA, ribosomes are released as monosomes. Some ribosomes are transferred to transfer-messenger RNA from the 3' end of mRNA. The monosomes thus produced are able to translate synthetic homopolymer but not natural mRNA with leader and canonical initiation signal. The pellet containing ribosomes appears to be responsible for rapid but reversible inhibition of most but not all of protein synthesis in vivo closely followed by decrease of cellular RNA and DNA synthesis.

[2004-11] Masuko, T. et al., Neurosci. Lett. 371, 30-33 (2004)

Dopamine has numerous functions in the brain and has been shown to modulate responses of N-methyl-D-aspartate (NMDA) receptors on thalamic and hippocampus neurons [N.G. Castro, M.C.F. de Mello, F.G. de Mello, Y. Aracava, Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393, Br. J. Pharmacol. 126 (1999) 1847-1855]. Thus, the effects of dopamine, serotonin, tyramine, epinephrine, norepinephrine, and octopamine on NMDA receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. Serotonin and tyramine, in addition to dopamine, were found to inhibit macroscopic currents at heteromeric NMDA receptors, but not AMPA (GluR1/GluR2) receptors. Epinephrine, norepinephrine and octopamine also weakly inhibited macroscopic currents at NR1/NR2A and NR1/NR2B receptors. The inhibitory effects of these monoamines became prominent at -100mV comparing those at -20mV. Mutations at NR1 N616, NR2B N615, and NR2B N616, but not at NR1 W563 and NR1 N650, reduced the inhibitory effects by monoamines. These results indicate that these monoamines directly act on the narrowest region of channel pore.

[2004-12] Higashi, K. et al., J. Biochem. 136, 533-539 (2004)

Following the report that agmatine has an anti-proliferative effect on cell growth through induction of antizyme [Satriano et al. (1998) J. Biol. Chem. 273, 15313-15316], we examined the effects of 16 different diamines on cell growth. Many diamines had little or no effect on cell growth, but agmatine and 1,6-hexanediamine had anti-proliferative effects, with agmatine having the strongest effect. Inhibition of cell growth occurred after 2 days, and inhibitory effects paralleled the degree of antizyme induction. Decreased spermine levels indicated that induction of spermidine/spermine N1-acetyltransferase was also involved in the inhibition of cell growth by agmatine and 1,6-hexanediamine. The frameshift efficiency (ratio of antizyme synthesis with or without frameshift) measured in a rabbit reticulocyte cell-free system was also increased by 1,3-propanediamine and cis-1,4-cyclohexanediamine in addition to agmatine and 1,6-hexanediamine. However, the intracellular levels of 1,3-propanediamine and cis-1,4-cyclohexanediamine were low when these compounds were added to the cell-culture medium. Other diamines had no effect on cell growth or frameshift efficiency. The results suggest that the presence of two amino-groups separated by an appropriate distance is important for the enhancement of frameshifting by diamines.

[2004-13] Takayama, K. et al., Biochem. Biophys. Res. Commun. 325, 1367-1375 (2004)

We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [35S]procaspase-9, but not [35S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos..