The pufferfish skeleton lacks ribs and pelvic fins, and has fused bones in the cranium and jaw. It has been hypothesized that this secondarily simplified pufferfish morphology is due to reduced complexity of the pufferfish Hox complexes. To test this hypothesis, we determined the genomic structure of Hox clusters in the Southern pufferfish Spheroides nephelus and interrogated genomic databases for the Japanese pufferfish Takifugu rubripes (fugu). Both species have at least seven Hox clusters, including two copies of Hoxb and Hoxd clusters, a single Hoxc cluster, and at least two Hoxa clusters, with a portion of a third Hoxa cluster in fugu. Results support genome duplication before divergence of zebrafish and pufferfish lineages, followed by loss of a Hoxc cluster in the pufferfish lineage and loss of a Hoxd cluster in the zebrafish lineage. Comparative analysis shows that duplicate genes continued to be lost for hundreds of millions of years, contrary to predictions for the permanent preservation of gene duplicates. Gene expression analysis in fugu embryos by in situ hybridization revealed evolutionary change in gene expression as predicted by the duplication-degeneration-complementation model. These experiments rule out the hypothesis that the simplified pufferfish body plan is due to reduction in Hox cluster complexity, and support the notion that genome duplication contributed to the radiation of teleosts into half of all vertebrate species by increasing developmental diversification of duplicate genes in daughter lineages.
Indiplon (NBI 34060) is a novel pyrazolopyrimidine currently in development for the treatment of insomnia. We have previously shown that indiplon exhibits high-affinity binding to native GABA A receptors from rat brain and acts as a positive allosteric modulator of GABA A receptor currents in cultured rat neurons (Sullivan et al., 2004). In this study, we examined the GABA A receptor ␣ subunit selectivity of indiplon using electrophysiological techniques to record GABA-activated chloride currents from recombinant rodent GABA A receptors expressed in human embryonic kidney 293 cells. Indiplon potentiated the GABA-activated chloride current in recombinant GABA A receptors in a dose-dependent and reversible manner and was approximately 10-fold selective for ␣1 subunit-containing receptors over GABA A receptors containing ␣2, ␣3, or ␣5 subunits. The EC 50 values were 2.6, 24, 60, and 77 nM for ␣12␥2, ␣22␥2, ␣33␥2, and ␣52␥2 receptors, respectively. Indiplon was approximately 10 times more potent than zolpidem and zopiclone and Ͼ100 times more potent than zaleplon. Moreover, indiplon, up to 1 M, did not potentiate GABA A receptors composed of ␣42␥2 and ␣62␥2 subunits. This mechanism of action is proposed to underlie the sedative-hypnotic effects of indiplon in animals and humans.GABA is the major inhibitory neurotransmitter in the mammalian central nervous system (Macdonald and Olsen, 1994). Drugs that enhance inhibitory neurotransmission include benzodiazepines, barbiturates, and general anesthetics and are used therapeutically as sedative-hypnotics, anxiolytics, antiepileptics, muscle relaxants, and anesthetics. These drugs target the GABA A receptor, an ion channel that selectively passes chloride when gated by the binding of GABA. Chloride influx serves to hyperpolarize or stabilize a negative resting membrane potential, making the neuron resistant to excitation.The GABA A receptor is a hetero-oligomeric complex composed of five transmembrane spanning subunits from sixteen different genes, ␣(1-6), (1-3), ␥(1-3), ␦, ⑀, , and (Barnard et al., 1998;Korpi et al., 2002;Whiting, 2003). In most neurons, two ␣ subunits, two  subunits, and one ␥ subunit form the typical GABA A receptor (Chang et al., 1996;Tretter et al., 1997). The ␦, ⑀, , and subunits have some reported selective functions but are not yet fully understood. Theoretically, there are thousands of possible subunit combinations, but a limited number of subtype combinations have been found in native systems with ␣12␥2, ␣23␥2, and ␣33␥2 being the most abundant (Whiting, 2003). The assembly of ␣, , and ␥ subunits is required to produce functional GABA A receptors that exhibit all of the pharmacological properties of native GABA A receptors. Benzodiazepine binding occurs at the interface between ␣ and ␥2 subunits (Wieland et al., 1992). GABA elicits chloride currents in recombinant GABA A receptors composed of only ␣ and  subunits, but these currents are not potentiated by benzodiazepines (Schofield et al., 1987).The diversity of subunits and...
Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock-out animals and antisense techniques have been used to study their specific roles. Here we used WAY-855, a GLT-1-preferring blocker, to assess the role of GLT-1 in rat hippocampus. GLT-1 was the most abundant transporter in the hippocampus at the mRNA level. According to [(3)H]-l-glutamate uptake data, GLT-1 was responsible for approximately 80% of the GLAST-, GLT-1-, and EAAC1-mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY-855 (100 microm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad-spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 microm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY-855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY-855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1-4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT-1 is insufficient to provoke glutamate build-up, leading to NMDA receptor-mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.
Reprogramming human somatic cells into pluripotent cells opens up new possibilities for transplantation therapy, the study of disease, and drug screening. In addition to somatic cell nuclear transfer, several approaches to reprogramming human cells have been reported: transduction of defined transcription factors to generate induced pluripotent stem cell (iPSC), human embryonic stem cell (hESC)-somatic cell fusion, and hESC cytoplast-somatic cell fusion or exposure to extracts of hESC. Here, we optimized techniques for hESC-human fibroblast fusion and enucleation and cytoplast fusion, and then compared the reprogramming efficiency between iPSC generation, cell-fusion and cytoplast-fusion. When compared with iPSC, hESC-fusion provided much faster and efficient reprogramming of somatic cells. The reprogramming required more than 4 weeks and the efficiency was less than 0.001% in iPSC generation, and it was less than 10 days and more than 0.005% in hESC-fusion. In addition, fusion yielded almost no partially reprogrammed cell colonies. However, the fused cells were tetraploid or aneuploid. hESC cytoplast fusion could initiate reprogramming but was never able to complete reprogramming. These data indicate that in cell fusion, as in nuclear transfer, reprogramming through direct introduction of a somatic nucleus into the environment of a pluripotent cell provides relatively efficient reprogramming. The findings also suggest that the nucleus of the host pluripotent cell may contain components that accelerate the reprogramming process.
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