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Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

The effects of benzyl-polyamines were studied at recombinant N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus laevis oocytes. A number of mono-, di- and tri-benzyl polyamines, having benzyl substitutions on the terminal or central amino groups, inhibited responses of NR1/NR2 receptors in oocytes voltage-clamped at -70 mV. Among the most potent compounds was N1,N4, N8-tri-benzyl-spermidine (TB-3-4), which had an IC50 value of 0.2 microM. TB-3-4 was approximately 40-fold more potent at NR1/NR2A and NR1/NR2B receptors than at NR1/NR2C or NR1/NR2D receptors. Block by TB-3-4 was strongly voltage dependent. Using voltage ramps analyzed by the Woodhull model of voltage-dependent channel block, TB-3-4 was found to have a Kd(0) value of 5 microM and a zdelta value of 1.41 at NR1/NR2B channels, whereas the affinity of binding [Kd(0) = 250 microM] but not the degree of voltage-dependence (zdelta = 1.43) was much lower at NR1/NR2D channels. At a concentration of 10 microM, TB-3-4 had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors expressed from the GluR1 subunit, indicating that TB-3-4 is a selective NMDA antagonist. TB-3-4 did not permeate wild-type NMDA channels but could easily permeate channels containing an N616G mutation in the NR1 subunit. This mutation is presumed to increase the size of the narrowest constriction of the NMDA channel, thus allowing passage of TB-3-4. Benzyl-polyamines such as TB-3-4 represent a structurally novel class of NMDA receptor channel blockers.

Glutamate and glycine are coagonists that act at distinct sites to activate N-methyl-D-aspartate (NMDA) receptors. In the NR1 subunit of the NMDA receptor, mutation of D732 to glutamate (D732E), asparagine (D732N), alanine (D732A), or glycine (D732G) reduced the potency of glycine by4000-fold, but these mutations had no effect on sensitivity to glutamate. Mutations at NR1(D732) also changed sensitivity to the glycine-site agonists D-serine and D-alanine, reducing the potencies and, in some cases, the efficacies of these compounds. Thus, D-serine was a full agonist at the glycine site of receptors containing NR1(D732N) and NR1(D732A), a partial agonist at receptors containing NR1(D732G), and a competitive antagonist at receptors containing NR1(D732). Mutations at NR1(D732) had no effect or produced an increase in sensitivity to the glycine-site antagonists 6,7-dichloroquinoxaline-2,3-dione and 5,7-dichlorokynurenic acid. These mutations did not affect the reversal potential, voltage-dependent block by extracellular Mg2+, block by ifenprodil, or stimulation by spermine at NR1/NR2B receptors. NR2 subunits containing mutations at NR2A(D731) and NR2B(D732), which correspond to NR1(D732), did not produce functional receptors when coexpressed with NR1. Residue D732 in NR1 may be close to a glycine binding site on the NMDA receptor and may directly affect the properties of this site or be critical for coupling of glycine binding to channel activation.

The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.

Effects of 1,15-bis(ethylamino)-4,8,12-triazapentadecane (BE3333), the least toxic bis(ethyl)pentaamine, on the growth of tumor cells were studied in in vitro systems and with tumor xenografts in mice. BE3333 suppressed ornithine decarboxylase and S-adenosylmethionine decarboxylase, induced spermidine/spermine N1-acetyltransferase, and thus decreased the amount of polyamines. BE3333 accumulated in cells at a concentration 3-5-fold that of spermine in control cells through the polyamine transport system. The accumulated BE3333 inhibited protein synthesis, especially mitochondrial protein synthesis, and decreased the amount of ATP. The inhibition of protein synthesis was correlated with the subsequent inhibition of cell growth. BE3333 showed inhibitory effects in in vitro systems against the growth of mouse FM3A mammary carcinoma cells, human SW480 and SW620 colon tumor cells, Lu-65A and A549 lung tumor cells, MCF-7 breast tumor cells, and MALME-3M and A375 melanoma cells at a range of 0.5-10 microM. Intravenous (30 mg/kg) or i.p. (50 mg/kg) daily injections of BE3333 for 5 or 7 days greatly suppressed the growth of human colon tumor SW620 xenotransplanted into nude mice. Similar antitumor activity was obtained with continuous infusion of BE3333 into the peritoneal cavity (80 mg/kg), but not with p.o. administration (200 mg/kg). BE3333 also showed inhibitory effects against the growth of lung tumors (Lu-65, Lx-1, Lc-1, and Lu-61), stomach tumors (Sc-6 and St-15), and melanoma (SEKI) xenotransplanted into nude mice. The results indicate that BE3333 is effective against both rapid- and slow-growing tumors, with reasonable short-term host toxicity.

The effect of the 5'-untranslated region of ornithine decarboxylase (ODC) mRNA and of spermidine on the translation of ODC mRNA was studied in a rabbit reticulocyte cell-free system. The ODC mRNAs, possessing different sizes of the 5'-untranslated region and named mODC241, mODC188, mODC85, and mODC24 according to the number of nucleotides in the 5'-untranslated region, were synthesized by in vitro transcription techniques through the use of plasmids containing various sizes of mouse ODC cDNA. As the size of the 5'-untranslated region of ODC mRNA increased, the efficiency of the translation decreased and the degree of stimulation of the translation by 0.2 mM spermidine increased. The inhibition of ODC mRNA translation by high concentrations of spermidine (0.6-1 mM) also increased with an increase in the size of the 5'-untranslated region. The spermidine effects were increased greatly when the size of the 5'-untranslated region of ODC mRNA was increased from 85 to 188 nucleotides. These results suggest that the nucleotides 70-220 upstream from the initiator AUG are involved in the decrease in translational efficiency. In addition, at least the nucleotides in the 70-170 region upstream from the initiator AUG are important in the strong stimulation of ODC synthesis by low spermidine concentrations and in the inhibition of ODC synthesis at high spermidine concentrations.

A hallmark feature of N-methyl-D-aspartate (NMDA) receptors is their voltage-dependent block by extracellular Mg2+. The structural basis for Mg2+ block is not fully understood. Although asparagine residues in the pore-forming M2 regions of NR1 and NR2 subunits influence Mg2+ block, it has been speculated that additional residues are likely to be involved. Here, we report the unexpected finding that a tryptophan residue in the M2 region of NR2 subunits controls Mg2+ block. An NR2B(W607L) mutation abolished block and greatly increased permeation of extracellular Mg2+. A similar effect was seen with a mutation at the equivalent residue in NR2A but not with mutations at the equivalent residue or adjacent residues in NR1. In NR2B, mutations that changed NR2B(W607) to asparagine (W607N) or alanine (W607A) also greatly reduced Mg2+ block, whereas mutations that changed W607 to the aromatic residues tyrosine (W607Y) or phenylalanine (W607F) had little or no effect on Mg2+ block. Furthermore, the W607L, W607N, and W607A mutants, but not the W607Y and W607F mutants, decreased Ba2+ permeability of NMDA channels. Thus, residue NR2B(W607) may be involved in binding of divalent cations, in particular Mg2+, through a cation-pi interaction with the electron-rich aromatic ring of the tryptophan. We previously suggested that NR2B(W607) may contribute to the narrow constriction of the NMDA channel. A model is now proposed in which the M2 loop of NR2B is folded in such a way that NR2B(W607) is positioned at the narrow constriction, at a level similar to NR2B(N616) and NR1(N616), with these three residues forming a binding site for Mg2+.

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.

Present experimental data show that the synthesis of ribosomal protein S1 and PI protein was stimulated greatly by polyamines at the early stage after addition of putrescine in polyamine-requiring mutants of E. coli. No macromolecular synthesis was stimulated at this stage. Polyamine stimulation of the synthesis of these proteins probably plays an important role for cell growth. In polyamine-deficient bovine lymphocytes, protein synthesis became perturbed before RNA and DNA synthesis. Among enzymes concerned with DNA replication, thymidine kinase activity was most strongly influenced by polyamines. The activity in polyamine-deficient cells was only 7% of the level in normal cells. Judging from the amount of thymidine kinase mRNA and its distribution in polysomes, it was concluded that polyamines mainly regulate the synthesis of thymidine kinase at the level of initiation of protein synthesis. A polyamine-free protein synthetic system, established from components of rabbit reticulocytes, consisted of globin mRNA, salt-washed ribosomes, partially purified initiation factors, and pH 5 enzymes. Spermidine added to this system not only lowered the optimal magnesium concentration required for globin synthesis, but it also stimulated the globin synthesis 8- to 10-fold. The optimal spermidine concentration was 0.4 to 0.6 mM, a concentration similar to that in intact rabbit reticulocytes. The ratio of alpha to beta globin chains synthesized in the presence of spermidine and Mg2+ was approximately 1.0, while the ratio in the presence of only Mg2+ was approximately 1.5. The results strongly suggest that polyamines play an important role in rabbit reticulocyte protein synthesis.

Purpose Normal tension glaucoma (NTG) is a subtype of glaucoma in which intraocular pressure is within the statistically normal range. NTG may be associated with an immune disorder. The aim of this study was to determine whether specific alleles in the human leukocyte antigen (HLA)-DRB1 and HLA-DQB1 genes correlated with NTG in Japanese patients. Methods We genotyped the HLA-DRB1 and HLA-DQB1 alleles in 113 Japanese patients with NTG and in 184 healthy Japanese control subjects using the polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP) Luminex method. We assessed the allelic diversity in patients and controls. Results There were no statistically significant differences in the allele frequency of HLADRB1 and HLA-DQB1 between NTG patients and control subjects, and no HLA-DRB1-HLA-DQB1 haplotypes demonstrated any significant association with NTG. Conclusions Our findings suggest that HLA-DRB1 and HLA-DQB1 polymorphisms have no significant effect on the development of NTG in Japanese patients.

The effects of aminoglycoside antibiotics on N-methyl-D-aspartate (NMDA) receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. A number of aminoglycosides were found to potentiate macroscopic currents at heteromeric NR1A/NR2B receptors, but not at NR1A/NR2A, NR1A/NR2C, NR1A/NR2D, or NR1B/NR2B receptors. The degree of potentiation had a rank order neomycin Bparomomycingentamicin Cgeneticinkanamycin Astreptomycin. Potentiation was not seen with kasugamycin and spectinomycin. The degree of stimulation paralleled the number of the amino groups in the aminoglycosides. The stimulatory effects of aminoglycosides were more pronounced at subsaturating concentrations of glycine and at acidic pH, similar to the stimulatory effects of spermine. We measured the effects of aminoglycosides at mutant NMDA receptors to determine which amino acid residues in NMDA receptor subunits are involved in stimulation. Mutations that reduced or abolished spermine stimulation also reduced stimulation by aminoglycosides. Several aminoglycosides produced a weak voltage-dependent block of NMDA receptors, but the degree of inhibition did not appear to correlate with the number of amino groups in the molecule. The results suggest that aminoglycosides having more than three amino groups have stimulatory effects that are mediated through the spermine-binding site on NMDA receptors.

To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.