Use of 2-[18F]fluoroethylazide for the Staudinger ligation – Preparation and characterisation of GABAA receptor binding 4-quinolones

The labelling reagent 2-[¹⁸F]fluoroethylazide was used in a traceless Staudinger ligation. This reaction was employed to obtain the GABA_A receptor binding 6-benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (2-[¹⁸F]fluoroethyl) amide. The radiotracer was prepared with a non-decay corrected radiochemical yield of 7%, a radiochemical purity >95% and a specific radioactivity of 0.9 GBq/μmol. The compound showed low brain penetration in normal rats. A series of fluoroalkyl 4-quinolone analogues with nanomolar to sub-nanomolar affinity for the GABA_A receptor has been prepared as well.     

Modelling competition and hybridization between native cutthroat trout and nonnative rainbow and hybrid trout

Native salmonid fish have been displaced worldwide by nonnatives through hybridization, competition, and predation, but the dynamics of these factors are poorly understood. We apply stochastic Lotka-Volterra models to the displacement of cutthroat trout by rainbow/hybrid trout in the Snake River, Idaho, USA. Cutthroat trout are susceptible to hybridization in the river but are reproductively isolated in tributaries via removal of migratory rainbow/hybrid spawners at weirs. Based on information-theoretic analysis, population data provide evidence that hybridization was the primary mechanism for cutthroat trout displacement in the first 17 years of the invasion. However, under some parameter values, the data provide evidence for a model in which interaction occurs among fish from both river and tributary subpopulations. This situation is likely to occur when tributary-spawned cutthroat trout out-migrate to the river as fry. The resulting competition with rainbow/hybrid trout can result in the extinction of cutthroat trout even when reproductive segregation is maintained.     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

Bim and Bcl-2 mutually affect the expression of the other in T cells

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.     

The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT-->CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our DeltamutT allele elevates the AT-->CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT-->CG transversions in our mutT(+) progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in DeltamutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the DeltamutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation.     

Design and synthesis of quinolin-2(1H)-one derivatives as potent CDK5 inhibitors

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine protein kinase and its deregulation is implicated in a number of neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and ischemic stroke. Using active site homology modeling between CDK5 and CDK2, we explored several different chemical series of potent CDK5 inhibitors. In this report, we describe the design, synthesis, and CDK5 inhibitory activities of quinolin-2(1H)-one derivatives.     

Accumulation of oxidatively generated DNA damage in the brain: a mechanism of neurotoxicity

Unrepaired or erroneously repaired DNA lesions drive genomic instability and contribute to cellular and organ decline. Since delayed neuropathologies are common in survivors of smoke inhalation injuries, we asked whether the integrity of brain DNA might be compromised by acute exposure to combustion smoke. Although many studies demonstrate that the brain is equipped to repair oxidatively damaged DNA, to date, the capacity for accurate DNA repair under conditions of disrupted oxygenation and oxidative stress has not been defined. We show that DNA adducts detectable by their ability to block PCR amplification form in the rat hippocampus after acute exposure to smoke. To identify the different types of adducts and to dissect their temporal formation and repair profiles in vivo in the brain, we used DNA-modifying enzymes to convert specific adducts into strand breaks prior to PCR amplification. Using this strategy, we detected formation of oxidative DNA adducts early on after smoke inhalation, while mismatched bases emerged at the later recovery times, potentially due to an erroneous DNA repair process. Erroneous repair can be mutagenic and because the initial smoke-induced oxidative damage to DNA is extensive, compromised fidelity of DNA repair may underlie neurotoxicity and contribute to delayed death of hippocampal neurons.     

Mitochondrial diversification of the Peromyscus mexicanus species group in Nuclear Central America: biogeographic and taxonomic implications

The mountain mice of the Peromyscus mexicanus group currently encompass six known species; however, the limits between species remain uncertain, with two considered monotypic and the other four having multiple associated subspecific names. Based on the most comprehensive sampling of the group throughout its distribution in Nuclear Central America, we used data of the mitochondrial cytochrome b gene to assess its genetic diversity, phylogeny, and main biogeographic and diversification patterns. Our mitochondrial phylogeny only partially reflects the current taxonomy of the group, in agreement with some of the taxonomically recognized species. Specifically, our phylogenetic results show that the group is highly structured, including four main clades with genetic distances ranging from 11 to 8.6%. A remarkable level of differentiation is found at a more local level, defined as 15 different lineages with high nucleotide and haplotype diversity (π = 0.068, h = 0.99), and with divergence and genetic distance values (p‐uncorrected = 9.9–2.4%; K2P = 10.8–3.0%) similar to values observed between species within Peromyscus. Accordingly, we propose that the reference name P. mexicanus is polyphyletic and should be restricted to the mountains of central Mexico west of the Isthmus of Tehuantepec. We suggest to limit the other five recognized specific names to equal number of lineages, as monophyletic, and to revalidate three junior synonyms: Peromyscus salvadorensis, P. nicaraguae and P. tropicalis. The Isthmus of Tehuantepec, the Motagua‐Polochic‐Jocotán fault system, the Maya Highlands and the Honduras Depression are examples of geographic features that are likely associated with the differentiation of main lineages. Some other lineages may represent candidate species, hence the need to review the taxonomic status of the entire P. mexicanus group.     

Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression

Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3′-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5.