The crystal structure of the oligonucleotide d(CGCAAATTO8GGCG), containing the chemically modified base 8-hydroxydeoxyguanine (O8G), has been determined at 2.5-A resolution and refined to a crystallographic R-factor of 16.8%. The B-type DNA helix contains standard Watson-Crick base pairs except at the mismatch sites, where O8G adopts a syn conformation and forms hydrogen bonds to adenine in the anti conformation. The thermodynamic stability of the duplex was found by UV melting techniques to be intermediate between the native oligonucleotide d(CGCAAATTTGCG) and an oligonucleotide containing A.G mispairs d(CGCAAATTGGCG). Comparison of the structure of the O8G(syn).A(anti) base pair with those of Watson-Crick base pairs has given a reason why O8G.A base pairs are not well repaired by DNA proofreading enzymes.

Background and Purpose-Grafts of MHP36 cells have previously been shown to reduce dysfunction after global ischemia in rats. To test their efficacy after focal ischemia, MHP36 cells were grafted 2 to 3 weeks after transient intraluminal middle cerebral artery occlusion (tMCAO) in rats. Methods-MHP36 cells were implanted into the hemisphere contralateral to the lesion, with 8 deposits of 3 L of cell suspension (25 000 cells per microliter). Sham grafted rats received equivalent volumes of vehicle. Three groups, sham-operated controls (nϭ11), MCAOϩsham grafts (nϭ10), and MCAOϩMHP36 grafts (nϭ11), were compared in 3 behavioral tests. Results-In the bilateral asymmetry test, MCAOϩMHP36 grafted rats exhibited neglect before grafting but subsequently showed no significant dysfunction, whereas MCAOϩsham grafted rats showed stable sensorimotor deficits over 18 weeks relative to controls. MCAOϩsham grafted rats demonstrated spontaneous motor asymmetry and increased rotational bias after injection of dopamine agonists. MCAOϩMHP36 and control groups exhibited no bias in either spontaneous or drug-induced rotation. In contrast to motor recovery, MCAOϩMHP36 grafted rats showed no improvement relative to MCAOϩsham grafted rats in spatial learning and memory in the water maze. MCAO produced large striatal and cortical cavitations in both occluded groups. Lesion volume was significantly reduced (PϽ0.05) in the MCAOϩMHP36 grafted group. The majority of MHP36 cells were identified within the intact grafted hemisphere. However, MHP36 cells were also seen in the cortex, striatum, and corpus callosum of the lesioned hemisphere. Conclusions-MHP36 cells may improve functional outcome after MCAO by assisting spontaneous reorganization in both the damaged and intact hemispheres.

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The self-complementary dodecanucleotide d [CGC(m6OAATTTGCG]2 (where m6G is 0'-methylguanine), which contains two m6G-T base pairs, has been analyzed by x-ray diffraction methods and the structure has been refined to a residual error ofR = 0.185 at 2.0-A resolution. The m6G-T mispair closely resembles a Watson-Crick base pair and there are very few structural differences between the m6G-T duplex and the native analogue. The similarity between the m6G-T base pair and a normal G-C base pair explains the failure of mismatch repair enzymes to recognize and remove this mutagenic lesion. A series of ultraviolet melting studies over a wide pH range on a related dodecamer indicate that the m6G-C mispair can exist in two conformations; one is a wobble pair and the other is a protonated Watson-Crick pair. The former, which predominates at physiological pH, will be removed by normal proofreading and repair enzymes, whereas the latter is likely to escape detection. Hence, the occasional occurrence of the protonated m6G-C base pair may explain why the presence of m6G in genomic DNA does not always give rise to a mutation.The initial stages of chemical carcinogenesis frequently involve the interaction ofgenotoxic agents with DNA to produce covalent modifications in the form of DNA adducts (1-3). An important example of this is the alkylation of the o6 position of guanine residues in DNA resulting from exposure to methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (4) and methyl methanesulfonate and N-methyl-N-nitrosourea (5). The presence of 06-methylguanine (m6G) constitutes a mutagenic lesion that is known to specifically induce G-C to A-T transition mutations (6) and it has been established that protooncogenes can be converted to oncogenes by such a process (7). Hence, the formation of the m6G-T base pair during replication can give rise to a carcinogenic lesion (8,9). In recent years, the biochemical processes involved in chemically induced carcinogenesis have been studied in considerable depth. However, to understand further the mechanisms of mutagenesis, it is necessary to analyze precisely the molecular details of the lesions produced when genotoxic agents interact with DNA. With this overall objective in mind, we have determined the structure of such a lesion, the m6G-T base pair in a B-DNA duplex. § MATERIALS AND METHODSAll oligonucleotides were synthesized by the solid-phase method on an ABI model 380B DNA synthesizer using cyanoethyl phosphoramidite monomers. For those containing m6G, the following protocol was observed: The 5'-dimethoxytrityl-N2-isobutyryl-06-methyldeoxyguanosine 3'-cyanoethyl phosphoramidite monomer was utilized to introduce 06-methyldeoxyguanosine and the fully assembled oligonucleotide was cleaved from the solid support and deprotected in a 5% solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in anhydrous methanol for 2 weeks at ambient temperature in an atmosphere of nitrogen (10). At no time was the oligonucleotide exposed to ammonia, as this can lead to the slow conver...

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo- 5,6,isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1-4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor, another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (K i of 4 and 5 M, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.Reduction of GABA-mediated inhibitory neurotransmission is associated with seizure activity and drugs that elevate synaptic GABA levels either by inhibition of GABA degradation or inhibition of high-affinity transport have been demonstrated to possess anticonvulsant activity (see Dalby, 2003;Sarup et al., 2003). For example, the GABA-transaminase inhibitor vigabatrin and the GABA-transport inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] are clinically effective antiepileptic drugs (for review and references, see Ben-Menachem, 2002;Kalviainen, 2002).Since the advent of cloning of several GABA transporters from different species including mouse, rat, and human, in-

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Convenient synthesis of rac-glycidaldehyde from rac-but-3-ene-1,2-diol and (R)-glycidaldehyde from D-mannitol are described. (R)-Glycidaldehyde (1) reacts with guanosine in water (pH 4-11, faster reaction at higher pH) to give initially 6(S)-hydroxy-7(S)-(hydroxymethyl)-3-(beta-D-ribofuranosyl)-5,6,7- trihydroimidazo[1,2-alpha]purin-9(3H)-one (7a) and 6(S),7(R)-dihydroxy-3-(beta-D-ribofuranosyl)-5,6,7,8- tetrahydropyrimido[1,2- alpha]purin-10(3H)-one (8a). The former decomposes to 7-(hydroxymethyl)-5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo[1,2- alpha]purine (3a), 5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo[1,2-alpha]purine (5a, 1,N2-ethenoguanosine), and formaldehyde, while the latter adduct is relatively stable. The position of the hydroxymethyl group on the imidazo ring of 7-(hydroxymethyl)-5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo-[1,2 - alpha]purine was proved by 13C NMR analysis of adducts derived from [1-15N]guanosine and [amino-15N]guanosine. At longer reaction times, the adduct 7,7'-methylenebis[5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo[1,2- alpha]purine (4a) is formed from guanosine and glycidaldehyde. The structure analysis of this adduct was also aided by 13C NMR analysis of the 15N-labeled adduct derived from [1-15N]guanosine. Analogous adducts were obtained from the reaction between glycidaldehyde and deoxyguanosine. Mechanisms of formation of the adducts from glycidaldehyde and guanosine/deoxyguanosine are proposed and supported by model studies with simple amines. The formaldehyde produced in the reactions described reacts with guanosine to give the known adduct N2-(hydroxymethyl)guanosine (9).

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