Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole

The rise of high mountain chains is widely seen as one of the factors driving rapid diversification of land plants and the formation of biodiversity hotspots. Supporting evidence was reported for the impact of the rapid rise of the Andean mountains but this hypothesis has so far been less explored for the impact of the “roof of the world”. The formation of the Himalaya, and especially the rise of the Qinghai–Tibetan Plateau in the recent 20 million years, altered the monsoon regimes that dominate the current climates of South East Asia. Here, we infer the hypothesis that the rise of Himalaya had a strong impact on the plant diversity in the biodiversity hotspot of the Southwest Chinese Mountains. Our analyses of the diversification pattern of the derived fern genus Lepisorus recovered evidence for changes in plant diversity that correlated with the strengthening of South East Asian monsoon. Southwest China or Southwest China and Japan was recovered as the putative area of origin of Lepisorus and enhancing monsoon regime were found to shape the early diversification of the genus as well as subsequent radiations during the late Miocene and Pliocene. We report new evidence for a coincidence of plant diversification and changes of the climate caused by the uplift of the Himalaya. These results are discussed in the context of the impact of incomplete taxon sampling, uncertainty of divergence time estimates, and limitations of current methods used to assess diversification rates.     

Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.     

Inhibition by bromoestrogens of the effects of estradiol on apomorphine-induced climbing behavior

The chronic administration of estrogens to mice or rats will result in antidopaminergic effects. Apomorphine-induced climbing behavior in mice, the result of direct stimulation of dopamine receptors in the striatal and mesolimbic regions, is a simple animal model for examining these antidopaminergic effects of estrogens. Bromoestrogens, inhibitors of catechol estrogen formation, have been utilized in order to examine the role of estrogen metabolism in dopaminergic antagonism. Mice were pretreated for 3 days with 2-bromoestradiol, 4-bromoestradiol, or 2,4-dibromoestradiol dibenzoates alone or in combination with estradiol benzoate prior to apomorphine administration. The haloestrogens did not alter the climbing-induced responses elicited by apomorphine, whereas estradiol benzoate clearly attentuated the actions of apomorphine. Furthermore, the bromoestradiol dibenzoates were effective in reversing the effects of estradiol benzoate when the two steroids (estradiol benzoate and a bromoestrogen dibenzoate) were administered simultaneously during pretreatment. Thus, the bromoestrogens are able to inhibit the antidopaminergic effects of estradiol exhibited in the apomorphine-induced mouse climbing model.     

Growth, compatible solute and salt accumulation of five mycorrhizal fungal species grown over a range of NaCl concentrations

The oil sand industry in northeastern Alberta produces vast areas of severely disturbed land. The sodicity of these anthropic soils is one of the principal constraints that impede their revegetation. Previous in vitro studies have shown that the ectomycorrhizal fungi Laccaria bicolor (Maire) Orton UAMH 8232 and Hebeloma crustuliniforme (Bull) Quel. UAMH 5247 have certain salt-resistant traits and thus are candidate species for the inoculation of tree seedlings to be outplanted on salt-affected soil. In this study, the in vitro development of these fungi was compared to that of three mycorrhizal fungi [Suillus tomentosus (Kauff.) Sing., Snell and Dick; Hymenoscyphus sp. and Phialocephala sp.] isolated from a sodic site created by Syncrude Canada Ltd. Their growth, osmotica and Na/Cl contents were assessed over a range (0, 50, 100, 200 mM) of NaCl concentrations. After 21 days, the two ascomycetes (Hymenoscyphus sp. and Phialocephala sp.) were shown to be more resistant to the NaCl treatments than the three basidiomycete species. Of the basidiomycetes, L. bicolor was the most sensitive to NaCl stress, while H. crustuliniforme showed greater water stress resistance, and the S. tomentosus isolate exhibited greater Na and Cl filtering capacities and had a better biomass yield over the NaCl gradient tested. Both ascomycetes used mechanisms other than carbohydrate accumulation to palliate NaCl stress. While the Hymenoscyphus isolate accumulated proline in response to NaCl treatments, the darker Phialocephala isolate may have used compounds such as melanin. The basidiomycete species accumulated mainly mannitol and/or proline in response to increasing concentrations of NaCl.     

Strain distribution in the layered wall of the esophagus.

The function of the esophagus is to move food by peristaltic motion, which is the result of the interaction of the tissue forces in the esophageal wall and the hydrodynamic forces in the food bolus. To understand the tissue forces in the esophagus, it is necessary to know the zero-stress state of the esophagus, and the stress-strain relationships of the tissues. This article is addressed to the first topic: the representation of zero-stress state of the esophagus by the states of zero stress-resultant and zero bending moment of the mucosa-submucosa and the muscle layers. It is shown that at the states of zero stress-resultant and zero bending moment, these two layers are not tubes of smaller radii but are open sectors whose shapes are approximately cylindrical and more or less circular. When the sectors are approximated by circular sectors, we measured their radii, opening angles, and average thickness around the circumference. Data on the radii, thickness-to-radius ratios, and the opening angles of these sectors are presented. Knowing the zero-stress state of these two layers, we can compute the strain distribution in the wall at any in vivo state, as well as the residual strain in the esophageal wall at the no-load state. The results of the in vivo states are compared to those obtained by a conventional approach, which treats the esophageal wall as a homogeneous material, and to another popular simplification, which ignores the residual strains completely. It is shown that the errors caused by the homogeneous wall assumption are relatively minor, but those caused by ignoring the residual strains completely are severe.     

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.     

Revealing Pathway Dynamics in Heart Diseases by Analyzing Multiple Differential Networks

Development of heart diseases is driven by dynamic changes in both the activity and connectivity of gene pathways. Understanding these dynamic events is critical for understanding pathogenic mechanisms and development of effective treatment. Currently, there is a lack of computational methods that enable analysis of multiple gene networks, each of which exhibits differential activity compared to the network of the baseline/healthy condition. We describe the iMDM algorithm to identify both unique and shared gene modules across multiple differential co-expression networks, termed M-DMs (multiple differential modules). We applied iMDM to a time-course RNA-Seq dataset generated using a murine heart failure model generated on two genotypes. We showed that iMDM achieves higher accuracy in inferring gene modules compared to using single or multiple co-expression networks. We found that condition-specific M-DMs exhibit differential activities, mediate different biological processes, and are enriched for genes with known cardiovascular phenotypes. By analyzing M-DMs that are present in multiple conditions, we revealed dynamic changes in pathway activity and connectivity across heart failure conditions. We further showed that module dynamics were correlated with the dynamics of disease phenotypes during the development of heart failure. Thus, pathway dynamics is a powerful measure for understanding pathogenesis. iMDM provides a principled way to dissect the dynamics of gene pathways and its relationship to the dynamics of disease phenotype. With the exponential growth of omics data, our method can aid in generating systems-level insights into disease progression.     

Tunicamycin-mediated depletion of insulin receptors in 3T3-L1 adipocytes.

Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3-L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anti-insulin receptor antibody on hexose uptake and metabolism.     

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IP_βII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IP_αII-N s) [1] on spectrin tetramer formation. The results showed that 3 IP_βII-C s were able to bind βII-C even in the presence of αII-N, and 4 IP_αII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.