Abstracts of papers (2003)

Last Update: 10/03/2006

Abstracts of papers (2003)

[2003-1] Masuko, T. et al., J. Neurochem. 84, 610-617 (2003)

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and snaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accmulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular sperimine, may function as neuromodulators in the brain.

[2003-2] Sakata, K. et al., Biochem. Soc. Trans. 31, 371-374 (2003)

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.

[2003-3] Williams, K. et al., J. Pharmacol. Exp. Ther. 305, 740-748 (2003)

The properties of delta2 receptors, which have homology to glutamate receptors but are not gated by glutamate, were studied using the constitutively active Lurcher mutant delta2(A654T) expressed in Xenopus oocytes. The macroscopic current through delta2(A654T) channels in voltage-clamped oocytes was defined as the difference between the holding current measured in the presence of extracellular Na(+) and that in the presence of the large impermeant cation N-methyl-d-glucamine. A-to-T mutations in the delta1 subunit and in NMDA (N-methyl-d-aspartate) receptor subunits, at positions equivalent to delta2(A654T), did not produce constitutively active channels. The current through delta2(A654T) channels was reduced by pentamidine and 9-tetrahydroaminoacridine, antagonists that also inhibit NR1/NR2B NMDA receptors but not AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. Block of delta2(A654T) currents by these two antagonists was incomplete and weakly voltage-dependent, in contrast to the block of NR1/NR2B receptors, which was complete and strongly voltage-dependent. Pentamidine inhibited a constitutively active NR1(T648A)/NR2B NMDA receptor in a manner similar to its inhibition of a glutamate-gated wild-type NMDA receptor, but different from its inhibition of constitutively active delta2(A654T) receptors. Currents gated by delta2(A654T) were sensitive to the extracellular pH, being smaller at acidic than at alkaline pH, with a pH IC(50) value of 7.47 and a maximum inhibition of 70%. It is concluded that delta2(A654T) channels have some properties in common with NMDA channels but also have characteristics that are different from these receptors. Compounds such as pentamidine may be useful for studies of native delta2 receptors.

[2003-4] Sakata, K. et al., Biochem. Biophys. Res. Commun. 305, 143-149 (2003)

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 [micro]M, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.

[2003-5] Low, C.-M. et al., Mol. Pharmacol. 63, 1212-1222 (2003)

Extracellular protons inhibit N-methyl-D-aspartate (NMDA) receptors with an IC50 value in the physiological pH range. To identify the molecular determinants of proton sensitivity, we used scanning mutagenesis of the NR1 subunit to search for residues that control proton inhibition of NMDA receptors. Homology modeling of the extracellular domains suggested that residues at which mutations perturbed pH sensitivity were localized in discrete regions. The majority of mutations that strongly affected proton sensitivity were clustered in the extracellular end of the second transmembrane domain (M3) and adjacent linker leading to the S2 portion of the glycine-binding domain of NR1. Mutations in NR2A confirmed that the analogous region controls the pH sensitivity of this subunit and also identified the linker region between the third transmembrane domain (M4) and the S2 portion of the NR2 glutamate binding domain as an additional determinant of proton sensitivity. One mutant receptor, NR1(A649C)/NR2A(A651T), showed a 145-fold reduction in the IC50 for protons (IC50, 17.3 [micro]M corresponding to pH 4.9). The M3-S2 linker region has been suggested to control NMDA receptor gating, leading to the hypothesis that the proton sensor and receptor gate may be structurally and functionally integrated.

[2003-6] Kai, M. et al., Cell Res. 13, 147-158 (2003)

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due to activation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of the apoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed into tadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpoles obtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cells of the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel at the early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection. We assume that apoptosis is executed in Xenopus early gastrulae as a "fail-safe" mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.

[2003-7] Ohmura-Hoshino, M. et al., Microbiol. Immunol. 47, 717-725 (2003)

A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum. Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA.