A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

Mycorrhizal dynamics under elevated CO2 and nitrogen fertilization in a warm temperate forest

We examined the response of mycorrhizal fungi to free-air CO2 enrichment (FACE) and nitrogen (N) fertilization in a warm temperate forest to better understand potential influences over plant nutrient uptake and soil carbon (C) storage. In particular, we hypothesized that mycorrhizal fungi and glomalin would become more prevalent under elevated CO2 but decrease under N fertilization. In addition, we predicted that N fertilization would mitigate any positive effects of elevated CO2 on mycorrhizal abundance. Overall, we observed a 14% increase in ectomycorrhizal (ECM) root colonization under CO2 enrichment, which implies that elevated CO2 results in greater C investments in these fungi. Arbuscular mycorrhizal (AM) hyphal length and glomalin stocks did not respond substantially to CO2 enrichment, and effects of CO2 on AM root colonization varied by date. Nitrogen effects on AM fungi were not consistent with our hypothesis, as we found an increase in AM colonization under N fertilization. Lastly, neither glomalin concentrations nor ECM colonization responded significantly to N fertilization or to an N-by-CO2 interaction. A longer duration of N fertilization may be required to detect effects on these parameters. © 2007 Springer Science+Business Media B.V

MCG+00-32-16: An Irregular Galaxy Close to the Lowest Redshift Absorber on the 3C 273 Line of Sight

MCG+00-32-16 is the galaxy closest in position-velocity space to the lowest redshift Ly$\alpha$ absorber along the line-of-sight to the quasar 3C 273. Its projected separation is 204 (d/19 Mpc) kpc, where d is the distance from the Milky Way to the galaxy, and the redshift difference is only 94 km/s; HI 1225+01 is slightly closer in projected separation to the absorber, but has a greater redshift difference. We present HI synthesis array mapping and CCD photometry in B and R for MCG+00-32-16. The HI disk is rotating in such a way that the side of the galaxy closer to the sight-line to the quasar has the larger velocity difference from the absorber. The absorber may be a ``failed dwarf'' member of a poor galaxy group of which MCG+00-32-16 and HI 1225+01 are the only members to have formed stars.Comment: 14 pages, 9 figures, accepted by Astrophysical Journa

A Study of Lyman-Alpha Quasar Absorbers in the Nearby Universe

Spectroscopy of ten quasars obtained with the Goddard High Resolution Spectrograph (GHRS) of the Hubble Space Telescope (HST) is presented. A clustering analysis reveals an excess of nearest neighbor line pairs on velocity scales of 250-750 km/s at a 95-98% confidence level. The hypothesis that the absorbers are randomly distributed in velocity space can be ruled out at the 99.8% confidence level. No two-point correlation power is detected (xi < 1 with 95% confidence). Lyman-alpha absorbers have correlation amplitudes on scales of 250-500 km/s at least 4-5 times smaller than the correlation amplitude of bright galaxies. A detailed comparison between absorbers in nearby galaxies is carried out on a limited subset of 11 Lyman- alpha absorbers where the galaxy sample in a large contiguous volume is complete to M_B = -16. Absorbers lie preferentially in regions of intermediate galaxy density but it is often not possible to uniquely assign a galaxy counterpart to an absorber. This sample provides no explicit support for the hypothesis that absorbers are preferentially associated with the halos of luminous galaxies. We have made a preliminary comparison of the absorption line properties and environments with the results of hydrodynamic simulations. The results suggest that the Lyman-alpha absorbers represent diffuse or shocked gas in the IGM that traces the cosmic web of large scale structure. (abridged)Comment: 36 pages of text, 15 figures, 4 tables, 36 file

Effective permittivity of random plasmonic composites

An effective-medium theory (EMT) is developed to predict the effective permittivity \epsilon_eff of dense random dispersions of high optical-conductivity metals such as Ag, Au and Cu. Dependence of \epsilon_eff on the volume fraction \phi, a microstructure parameter \kappa related to the static structure factor and particle radius a is studied. In the electrostatic limit, the upper and lower bounds of \kappa correspond to Maxwell-Garnett and Bruggeman EMTs respectively. Finite size effects are significant when |\beta^2(ka/n)^3| becomes O(1) where \beta, k, and n denote the nanoparticle polarizability, wavenumber and matrix refractive index respectively. The coupling between the particle and effective medium results in a red-shift in the resonance peak, a non-linear dependence of \epsilon_eff on \phi, and Fano resonance in \epsilon_eff.Comment: Manuscript submitted to J. Opt. Soc. Am. B. 33 page

Folding Pathways of Prion and Doppel

The relevance of various residue positions for the stability and the folding characteristics of the prion protein are investigated by using molecular dynamics simulations of models exploiting the topology of the native state. Highly significant correlations are found between the most relevant sites in our analysis and the single point mutations known to be associated with the arousal of the genetic forms of prion disease (caused by the conformational change from the cellular to the scrapie isoform). Considerable insight into the conformational change is provided by comparing the folding process of prion and doppel (a newly discovered protein) sharing very similar native state topology: the folding pathways of the former can be grouped in two main classes according to which tertiary structure contacts are formed first enroute to the native state. For the latter a single class of pathways leads to the native state. Our results are consistent and supportive of the recent experimental findings that doppel lacks the scrapie isoform and that such remarkably different behavior results from differences in the region containing the two $\beta-$strands and the intervening helix.Comment: 16 pages, 2 tables, 5 figure

Establishment of endomycorrhizal fungi on micropropagated teak (Tectona grandis L.f.)

No abstract available

Fuzzy-logic based inelastic displacement ratios of degrading RC structures

The existing classical methods for estimating the inelastic displacement ratios of reinforced concrete (RC) structures subjected to seismic excitation are built upon several assumptions that ignore the effect of uncertainties on the concerning phenomenon. Uncertainty techniques are more appropriate to modeling such phenomenon that inherits impreciseness. This research presents a new method predicting the inelastic displacement ratio of moderately degrading RC structures subjected to earthquake loading using expert systems such as fuzzy logic approach. A well-defined degrading model was used to conduct the dynamic analyses. A total of 300 earthquake motions recorded on firm sites, including recent ones from Japan and New Zealand, with magnitudes greater than 5 and peak ground acceleration (PGA) values greater than 0.08 g, were selected. These earthquake records were applied on five RC columns that were chosen among 255 tested columns based on their beam–column element parameters reported by the Pacific Earthquake Engineering Research Center (2003) [1]. A total of 96,000 dynamic analyses were conducted. The results from these analyses were used to develop the fuzzy inelastic displacement ratio model inheriting uncertainties in terms of strength reduction factor (R) and period of vibration (T). The performance evaluation of the new fuzzy logic model and four classical methods were investigated using different independent data sets. As a result, more accurate results were predicted using the new fuzzy logic model