Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli

Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were T transversions.     

Inhibition of cell proliferation in black‐capped chickadees suggests a role for neurogenesis in spatial learning

Following development, the avian brain continues to produce neurons throughout adulthood, which functionally integrate throughout the telencephalon, including the hippocampus. In food‐storing birds like the black‐capped chickadee (Poecile atricapillus), new neurons incorporated into the hippocampus are hypothesized to play a role in spatial learning. Previous results on the relation between hippocampal neurogenesis and spatial learning, however, are correlational. In this study, we experimentally suppressed hippocampal neuronal recruitment and tested for subsequent effects on spatial learning in adult chickadees. After chickadees exhibited significant learning, we treated birds with daily injections of either saline or methylazoxymethanol (MAM), a toxin that suppresses cell proliferation in the brain and monitored subsequent spatial learning. MAM treatment significantly reduced cell proliferation around the lateral ventricles and neuronal recruitment in the hippocampus, measured using the cell birth marker bromodeoxyuridine. MAM‐treated birds performed significantly worse than controls on the spatial learning task 12 days following the initiation of MAM treatment, a time when new neurons would begin functionally integrating into the hippocampus. This difference in learning, however, was limited to a single trial. MAM treatment did not affect any measure of body condition, suggesting learning impairments were not a product of non‐specific adverse effects of MAM. This is the first evidence of a potential causal link between hippocampal neurogenesis and spatial learning in birds. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1002–1010, 2014     

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.     

Sympathetic neural responses to increased osmolality in humans

The purpose of this study was to examine the relationship between osmolality and efferent sympathetic outflow in humans. We hypothesized that increased plasma osmolality would be associated with increases in directly measured sympathetic outflow. Muscle sympathetic outflow was successfully recorded in eight healthy subjects during a 60-min intravenous hypertonic saline infusion (HSI; 3% NaCl) on one day and during a 60-min intravenous isotonic saline (ISO) infusion (0.9% NaCl) on a different day. The HSI provides an osmotic and volume stimulus, whereas the ISO infusion provides a volume-only stimulus. Muscle sympathetic nerve activity was quantified using the technique of peroneal microneurography. Plasma osmolality increased during the HSI but not during the ISO infusion (ANOVA, P < 0.05). Sympathetic outflow differed between the trials (ANOVA, P < 0.05); during the HSI burst, frequency initially increased from 14.6 +/- 2.5 to 18.1 +/- 1.9 bursts/min; during the ISO infusion, burst frequency initially declined from 14.7 +/- 2.5 to 12.0 +/- 2.1 bursts/min. Plasma norepinephrine concentration was greater at the end of the HSI compared with the end of the ISO infusion (HSI: 297 +/- 64 vs. ISO: 202 +/- 49 pg/ml; ANOVA, P < 0.05). We conclude that HSI-induced increases in plasma osmolality are associated with increases in sympathetic activity in humans.     

SWAN pathway-network identification of common aneuploidy-based oncogenic drivers

Haploinsufficiency drives Darwinian evolution. Siblings, while alike in many aspects, differ due to monoallelic differences inherited from each parent. In cancer, solid tumors exhibit aneuploid genetics resulting in hundreds to thousands of monoallelic gene-level copy-number alterations (CNAs) in each tumor. Aneuploidy patterns are heterogeneous, posing a challenge to identify drivers in this high-noise genetic environment. Here, we developed Shifted Weighted Annotation Network (SWAN) analysis to assess biology impacted by cumulative monoallelic changes. SWAN enables an integrated pathway-network analysis of CNAs, RNA expression, and mutations via a simple web platform. SWAN is optimized to best prioritize known and novel tumor suppressors and oncogenes, thereby identifying drivers and potential druggable vulnerabilities within cancer CNAs. Protein homeostasis, phospholipid dephosphorylation, and ion transport pathways are commonly suppressed. An atlas of CNA pathways altered in each cancer type is released. These CNA network shifts highlight new, attractive targets to exploit in solid tumors.     

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.     

Linkage of the multiple endocrine neoplasia type 2B gene (MEN2B) to chromosome 10 markers linked to MEN2A

The syndrome of multiple endocrine neoplasia type 2B (MEN 2B) resembles that of MEN 2A in that both include medullary carcinoma of the thyroid, pheochromocytoma, and autosomal dominant inheritance, but is distinct in that MEN 2B patients have neuromas of the mucous membranes. MEN2A has been linked to RBP3, D10S5, FNRB, D10S15, and D10Z1 near the centromere of chromosome 10. We examined linkage between MEN2B and RFLPs on chromosome 10 in all available members in two or three generations of 14 kindreds. The centromere marker D10Z1 was linked to MEN2B with a peak lod score of 5.42 at theta = 0.02. One possible recombinant was observed between D10Z1 and MEN2B. Multipoint analysis of RFLPs at FNRB, D10Z1, RBP3, and D10S15 gave a peak lod score of 7.12 at the midpoint between D10Z1 and RBP3 on the long arm (band q11). The most likely gene order FNRB-D10Z1-MEN2B was 27 times more likely than MEN2B-FNRB-D10Z1 and 31/2 times more likely than FNRB-MEN2B-D10Z1. Additional data will be required to establish the order of these loci with confidence.