Canadian oil sands tailings are predominately sodic residues contaminated by hydrocarbons such as naphthenic acids. These conditions are harsh for plant development. In this study, we evaluated the effect of inoculating roots of Alnus viridis ssp. crispa and Alnus incana ssp. rugosa with ectomycorrhizal fungi in the presence of tailings compounds. Seedlings were inoculated with 7 different strains of Paxillus involutus and Alpova diplophloeus and were grown under different treatments of NaCl, Na2SO4, and naphthenic acids in a growth chamber. Afterwards, seedling survival, height, dry biomass, leaf necrosis, and root mycorrhization rate were measured. Paxillus involutus Mai was the most successful strain in enhancing alder survival, health, and growth. Seedlings inoculated with this strain displayed a 25% increase in survival rate, 2-fold greater biomass, and 2-fold less leaf necrosis compared with controls. Contrary to our expectations, A. diplophloeus was not as effective as P. involutus in improving seedling fitness, likely because it did not form ectomycorrhizae on roots of either alder species. High intraspecific variation characterized strains of P. involutus in their ability to stimulate alder height and growth and to minimize leaf necrosis. We conclude that in vivo selection under bipartite symbiotic conditions is essential to select effective strains that will be of use for the revegetation and reclamation of derelict lands.

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

Spinocerebellar ataxia type 1 is an autosomal dominant cerebellar ataxia associated with the expansion of a polyglutamine tract within the ataxin-1 (ATXN1) protein. Recent studies suggest that understanding the normal function of ATXN1 in cellular processes is essential to decipher the pathogenesis mechanisms in spinocerebellar ataxia type 1. We found an alternative translation initiation ATG codon in the +3 reading frame of human ATXN1 starting 30 nucleotides downstream of the initiation codon for ATXN1 and ending at nucleotide 587. This novel overlapping open reading frame (ORF) encodes a 21-kDa polypeptide termed Alt-ATXN1 (Alternative ATXN1) with a completely different amino acid sequence from ATXN1. We introduced a hemagglutinin tag in-frame with Alt-ATXN1 in ATXN1 cDNA and showed in cell culture the co-expression of both ATXN1 and Alt-ATXN1. Remarkably, Alt-ATXN1 colocalized and interacted with ATXN1 in nuclear inclusions. In contrast, in the absence of ATXN1 expression, Alt-ATXN1 displays a homogenous nucleoplasmic distribution. Alt-ATXN1 interacts with poly(A)(+) RNA, and its nuclear localization is dependent on RNA transcription. Polyclonal antibodies raised against Alt-ATXN1 confirmed the expression of Alt-ATXN1 in human cerebellum expressing ATXN1. These results demonstrate that human ATXN1 gene is a dual coding sequence and that ATXN1 interacts with and controls the subcellular distribution of Alt-ATXN1.

2,2′,4,4′‐tetrabromodiphenyl Ether (BDE‐47) is a flame‐retardant chemical appearing at increasing concentrations and frequency in the environment and human samples. A number of health effects of exposure to BDE‐47 have been observed, thyroid disruption being the most sensitive. Our objective was to examine BDE‐47 interaction with thyroid receptor beta (TRβ). We used a variety of approaches, including in vitro binding assays, luciferase reporter‐gene transcriptional assays, and analysis of expression of thyroid responsive genes in rat offspring exposed perinatally to BDE‐47. We found that BDE‐47 alone or in mixture with 2,2′,4,4′,5‐pentabromodiphenyl ether (BDE‐99), 2,2′,4,4′,6‐pentabromodiphenyl ether (BDE‐100), and 2,2′,4,4′,5,5′‐hexabromodiphenyl ether (BDE‐153) does not compete with [125I]T3 for TRβ‐binding even at 4000 fold higher concentrations. Also, BDE‐47 does not affect thyroid responsive genes through TRβ in in vitro studies of transcription regulation. A subset of thyroid responsive genes were significantly differentially expressed in liver and frontal lobe brain samples of exposed pups, however, the action of BDE‐47 was neither agonistic or antagonistic to that of thyroid hormone. We conclude that BDE‐47 does not interact directly with TRβ1 nor does it influence its transcriptional activity. Developmental exposure of rats to BDE‐47 leads to differential expression of thyroid responsive genes in liver and brain due to unknown mechanism. Copyright © 2010 John Wiley & Sons, Ltd.

Human activity, and in particular industrial activity, has altered natural environments. Here we present an experimental approach adapted to study the actinorhizal symbiosis in alder trees and shrubs submitted to abiotic stress. We measured the impact of exogenous nitrogen on the establishment of the alder symbiosis with Frankia sp., and its primary function; nitrogen fixation. Results showed our version of the growth pouch method was functional, and corroborated the gradual inhibition of symbiosis in the presence of increasing exogenous nitrogen concentrations. In mountain alder (Alnus viridis ssp. crispa) there was a gradual and suppressive effect of nitrogen on the relative number or root nodules, while in black alder (Alnus glutinosa) results suggested a threshold effect at 45 ppm N. Shoot to root biomass ratios were increased in the presence of the microsymbiont, and this effect was generally maintained even in the presence of heavy metals (As, Se or V). Alders and the actinorhizal symbiosis were not heavily affected by the presence of heavy metals, confirming potential applications in soil rehabilitation, however the distribution of metals in plant tissues sometimes changed when high levels of metals were present. A. glutinosa plants exposed to high levels of As significantly increased the allocation of As to roots (approximate to 90%), while those exposed to high levels of Se rose their aerial tissue Se allocation to roughly 86%. A. glutinosa plants exposed to high V levels did not change behavior: V was in all cases preferentially accumulated in underground tissues (>= 90%). Our results detail the use of a high-throughput approach to study the plasticity of the actinorhizal symbiosis in the presence of fluctuating nitrogen and metal conditions. These methods are transposable to numerous actinorhizal studies in both fundamental and applied research. (C) 2011 Elsevier B.V. All rights reserved.

BACKGROUND:Aggresomes are juxtanuclear inclusion bodies that have been proposed to represent a general cellular response to misfolded proteins in mammalian cells. Yet, why aggresomes are not a pathological characteristic of protein misfolding diseases is unclear. Here, we investigate if a misfolded protein inevitably forms aggresomes in mammalian cells.RESULTS:We show that a cytoplasmic form of the prion protein may form aggresomes or dispersed aggregates in different cell lines. In contrast to aggresomes, the formation of dispersed aggregates is insensitive to histone deacetylase 6 inhibitors and does not result in cytoskeleton rearrangements. Modulation of expression levels or proteasome inhibitors does not alter the formation of dispersed aggregates.CONCLUSION:Our results establish that aggresomes are not obligatory products of protein misfolding in vivo.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP C ) into an altered disease‐associated conformation generally designated PrP Sc , abnormal deposition of PrP Sc aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP C aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP C molecules may trigger its aggregation in neuronal cells. A chimeric PrP C composed of one (Fv1) or two (Fv2) modified FK506‐binding protein (Fv) fused with PrP C were created, and transfected in N2a cells. Similar to PrP C , Fv1‐PrP and Fv2‐PrP were glycosylated, displayed normal localization, and anti‐apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP C , both dimerization and oligomerization of PrP C resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non‐ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP C molecules may constitute a minimal and sufficient molecular event leading to PrP C aggregation and extracellular deposition.

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non‐ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non‐neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE(2)‐M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1–5‐µm inclusion bodies and were filled with electron‐dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild‐type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non‐neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.