Leaf-specific suppression of deoxyhypusine synthase in Arabidopsis thaliana enhances growth without negative pleiotropic effects

Deoxyhypusine synthase (DHS) mediates the first of two enzymatic reactions required for the post-translational activation of eukaryotic translation initiation factor 5A (eIF5A), which in turn is thought to facilitate translation of specific mRNAs. Analyses of GUS activity in transgenic Arabidopsis plants expressing the GUS reporter gene under regulation of the promoter for AtDHS revealed that the expression of DHS changes both spatially and temporally as development progresses. In particular, DHS is expressed not only in rosette leaves, but also in the anthers of developing flowers. To determine the role of DHS in leaves, transgenic plants in which DHS was selectively suppressed in rosettes of Arabidopsis plants were prepared. This was achieved by expressing AtDHS 3'-UTR cDNA as a transgene under regulation of the promoter for AtRbcS2, a gene encoding the small subunit of Rubisco. The dominant phenotypic traits of the DHS-suppressed plants proved to be a dramatic enhancement of both vegetative and reproductive growth. As well, the onset of leaf senescence in the DHS-suppressed plants was delayed by approximately 1 week, but there was no change in the timing of bolting. In addition, there was no evidence for the negative pleiotropic effects, including stunted reproductive growth and reduced seed yield, noted previously for transgenic plants in which DHS was constitutively suppressed. The results indicate that DHS plays a pivotal role in both growth and senescence.     

Phenology, distribution, and host specificity of Solenopsis invicta virus-1

Studies were conducted to examine the phenology, geographic distribution, and host specificity of the Solenopsis invicta virus-1 (SINV-1). Two genotypes examined, SINV-1 and -1A, exhibited similar seasonal prevalence patterns. Infection rates among colonies of S. invicta in Gainesville, Florida, were lowest from early winter (December) to early spring (April) increasing rapidly in late spring (May) and remaining high through August before declining again in the fall (September/October). Correlation analysis revealed a significant relationship between mean monthly temperature and SINV-1 (p<0.0005, r=0.82) and SINV-1A (p<0.0001, r=0.86) infection rates in S. invicta colonies. SINV-1 was widely distributed among S. invicta populations. The virus was detected in S. invicta from Argentina and from all U.S. states examined, with the exception of New Mexico. SINV-1 and -1A were also detected in other Solenopsis species. SINV-1 was detected in Solenopsis richteri and the S. invicta/richteri hybrid collected from northern Alabama and Solenopsis geminata from Florida. SINV-1A was detected in S. geminata and Solenopsis carolinensis in Florida and the S. invicta/richteri hybrid in Alabama. Of the 1989 arthropods collected from 6 pitfall trap experiments from Gainesville and Williston, Florida, none except S. invicta tested positive for SINV-1 or SINV-1A. SINV-1 did not appear to infect or replicate within Sf9 or Dm-2 cells in vitro. The number of SINV-1 genome copies did not significantly increase over the course of the experiment, nor were any cytopathic effects observed. Phylogenetic analyses of SINV-1/-1A nucleotide sequences indicated significant divergence between viruses collected from Argentina and the U.S.     

Antibody recognition of cell surface-associated NS1 triggers Fc-gamma receptor-mediated phagocytosis and clearance of West Nile Virus-infected cells

Previous studies have suggested that monoclonal antibodies (MAbs) to flavivirus nonstructural protein-1 (NS-1) protect against infection in mice through an Fc-gamma receptor-dependent pathway. To identify a specific mechanism, we evaluated the protective activity of anti-NS1 MAbs to WNV using mice and cells with deficiencies of specific Fc-gamma receptors. Our results suggest that only MAbs that recognize cell surface-associated NS1 trigger Fc-gamma receptor I- and/or IV-mediated phagocytosis and clearance of WNV-infected cells. These findings may be relevant for generating novel therapeutic MAbs or vaccines against flaviviruses that target the NS1 protein.     

Promotion of cancer cell migration: an insulin-like growth factor (IGF)-independent action of IGF-binding protein-6

A family of six high affinity IGF-binding proteins (IGFBPs 1-6) plays an important role in modulating IGF activities. Recent studies suggest that some IGFBPs may have IGF-independent effects, including induction of apoptosis and modulation of cell migration. However, very little is known about possible IGF-independent actions of IGFBP-6. We have generated a non-IGF-binding IGFBP-6 mutant by substituting Ala for four amino acid residues (Pro(93)/Leu(94)/Leu(97)/Leu(98)) in its N-domain IGF-binding site. A >10,000-fold loss of binding affinity for IGF-I and IGF-II was observed using charcoal solution binding assay, BIAcore biosensor, and ligand blotting. Wild-type and mutant IGFBP-6, as well as IGF-II, induced cell migration in RD rhabdomyosarcoma and LIM 1215 colon cancer cells. Cell migration was mediated by the C-domain of IGFBP-6. Transient p38 phosphorylation was observed in RD cells after treatment with IGFBP-6, whereas no change was seen in phospho-ERK1/2 levels. Phospho-JNK was not detected. IGFBP-6-induced cell migration was inhibited by SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of ERK1/2 MAPK activation. In contrast, SP600125, a JNK MAPK inhibitor, had no effect on migration. Knockdown of p38 MAPK using short interfering RNA blocked IGFBP-6-induced migration of RD cells. These results indicate that p38 MAPK is involved in IGFBP-6-induced IGF-independent RD cell migration.     

Effects of grazer identity on the probability of escapes by a canopy-forming macroalga

Through their grazing activities limpets have an important role in controlling macroalgal abundance and as a result the structure and dynamics of rocky shore assemblages. Using two congeneric limpet species, with different biogeographic distributions, and whose ranges are expected to alter with climatic warming, we separated the magnitude of their grazing activity over time and the subsequent consequence for macroalgal growth.The northern/boreal limpet, Patella vulgata (L.), consistently grazed more than the southern/lusitanian limpet, P. depressa (Pennant), particularly during spring and summer when P. depressa was reproductively active. Individuals of Fucus vesiculosus (L.) that settled during this time were able to grow to a size where they escaped the grazing activities of P. depressa, resulting in mature F. vesiculosus being present in all P. depressa treatments. In contrast, P. vulgata, which was not reproductively active during this period, exhibited no reduction in its grazing activity and prevented macroalgae from growing in experimental treatments. It therefore appears that P. vulgata has a stronger role, than P. depressa, in controlling macroalgal abundance on shores of southwest Britain.We present a conceptual model highlighting the direct and indirect interactions between these two limpet species and F. vesiculosis. If as predicted, under current warming scenarios, P. depressa becomes the dominant limpet on shores of southwest Britain there will be subsequent changes in rocky shore community structure and ecosystem functioning. Our research emphasises that even closely related species with similar ecological niches can exhibit different behaviours that fundamentally alter their biological interactions with other organisms leading to idiosyncratic responses to predicted changes in climate.     

Targeted disruption of the murine retinal dehydrogenase gene Rdh12 does not limit visual cycle function

RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark- and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12(-/-) animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.     

Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis

Fanconi anemia (FA) is a chromosomal instability disorder in which DNA-damage processing defects are reported for translesion synthesis (TLS), non-homologous end joining (NHEJ) and homologous recombination (HR; both increased and decreased). To reconcile these diverse findings, we compared spontaneous mutagenesis in FA and HR mutants of hamster CHO cells. In the fancg mutant we find a reduced mutation rate accompanied by an increased proportion of deletions within the hprt gene. Moreover, in fancg cells gene amplification at the CAD and dhfr loci is elevated, another manifestation of inappropriate processing of damage during DNA replication. In contrast, the rad51d HR mutant has a greatly elevated rate of hprt mutations, >85% of which are deletions. Our analysis supports the concept that HR faithfully restores broken replication forks, whereas the FA pathway acts more globally to ensure chromosome stability by promoting efficient end joining of replication-derived breaks, as well as TLS and HR.