669 research outputs found
Practical Spectrophotometric Assay for the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-L,L-Diaminopimelic Acid Desuccinylase, a Potential Antibiotic Target
A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] ΌM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] ΌM, phenylboronic acid (IC50 = 316 [± 23.6] ΌM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] ΌM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics
RNA-seq analysis of ageing human retinal pigment epithelium: Unexpected up-regulation of visual cycle gene transcription
Ageing presents adverse effects on the retina and is the primary risk factor for ageârelated macular degeneration (AMD). We report the first RNAâseq analysis of ageârelated transcriptional changes in the human retinal pigment epithelium (RPE), the primary site of AMD pathogenesis. Whole transcriptome sequencing of RPE from human donors ranging in age from 31 to 93 reveals that ageing is associated with increasing transcription of main RPEâassociated visual cycle genes (including LRAT, RPE65, RDH5, RDH10, RDH11; pathway enrichment BHâadjusted PÂ =Â 4.6Â ĂÂ 10(â6)). This positive correlation is replicated in an independent set of 28 donors and a microarray dataset of 50 donors previously published. LRAT expression is positively regulated by retinoid byâproducts of the visual cycle (A2E and allâtransâretinal) involving modulation by retinoic acid receptor alpha transcription factor. The results substantiate a novel ageârelated positive feedback mechanism between accumulation of retinoid byâproducts in the RPE and the upâregulation of visual cycle genes
CFH, C3 and ARMS2 Are Significant Risk Loci for Susceptibility but Not for Disease Progression of Geographic Atrophy Due to AMD
Age-related macular degeneration (AMD) is a prevalent cause of blindness in Western societies. Variants in the genes encoding complement factor H (CFH), complement component 3 (C3) and age-related maculopathy susceptibility 2 (ARMS2) have repeatedly been shown to confer significant risks for AMD; however, their role in disease progression and thus their potential relevance for interventional therapeutic approaches remains unknown. Here, we analyzed association between variants in CFH, C3 and ARMS2 and disease progression of geographic atrophy (GA) due to AMD. A quantitative phenotype of disease progression was computed based on longitudinal observations by fundus autofluorescence imaging. In a subset of 99 cases with pure bilateral GA, variants in CFH (Y402H), C3 (R102G), and ARMS2 (A69S) are associated with disease (P = 1.6x10(-9), 3.2x10(-3), and P = 2.6x10(-12), respectively) when compared to 612 unrelated healthy control individuals. In cases, median progression rate of GA over a mean follow-up period of 3.0 years was 1.61 mm(2)/year with high concordance between fellow eyes. No association between the progression rate and any of the genetic risk variants at the three loci was observed (P>0.13). This study confirms that variants at CFH, C3, and ARMS2 confer significant risks for GA due to AMD. In contrast, our data indicate no association of these variants with disease progression which may have important implications for future treatment strategies. Other, as yet unknown susceptibilities may influence disease progression
Quasi-Newtonian dust cosmologies
Exact dynamical equations for a generic dust matter source field in a
cosmological context are formulated with respect to a non-comoving
Newtonian-like timelike reference congruence and investigated for internal
consistency. On the basis of a lapse function (the relativistic
acceleration scalar potential) which evolves along the reference congruence
according to (), we find that
consistency of the quasi-Newtonian dynamical equations is not attained at the
first derivative level. We then proceed to show that a self-consistent set can
be obtained by linearising the dynamical equations about a (non-comoving) FLRW
background. In this case, on properly accounting for the first-order momentum
density relating to the non-relativistic peculiar motion of the matter,
additional source terms arise in the evolution and constraint equations
describing small-amplitude energy density fluctuations that do not appear in
similar gravitational instability scenarios in the standard literature.Comment: 25 pages, LaTeX 2.09 (10pt), to appear in Classical and Quantum
Gravity, Vol. 15 (1998
PIP5KIÎČ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells
Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, ÎČ or Îł). PIP5KIÎČ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIÎČ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIÎČ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIÎČ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al
The Cosmological Constant
This is a review of the physics and cosmology of the cosmological constant.
Focusing on recent developments, I present a pedagogical overview of cosmology
in the presence of a cosmological constant, observational constraints on its
magnitude, and the physics of a small (and potentially nonzero) vacuum energy.Comment: 50 pages. Submitted to Living Reviews in Relativity
(http://www.livingreviews.org/), December 199
The Hubble Constant
I review the current state of determinations of the Hubble constant, which
gives the length scale of the Universe by relating the expansion velocity of
objects to their distance. There are two broad categories of measurements. The
first uses individual astrophysical objects which have some property that
allows their intrinsic luminosity or size to be determined, or allows the
determination of their distance by geometric means. The second category
comprises the use of all-sky cosmic microwave background, or correlations
between large samples of galaxies, to determine information about the geometry
of the Universe and hence the Hubble constant, typically in a combination with
other cosmological parameters. Many, but not all, object-based measurements
give values of around 72-74km/s/Mpc , with typical errors of 2-3km/s/Mpc.
This is in mild discrepancy with CMB-based measurements, in particular those
from the Planck satellite, which give values of 67-68km/s/Mpc and typical
errors of 1-2km/s/Mpc. The size of the remaining systematics indicate that
accuracy rather than precision is the remaining problem in a good determination
of the Hubble constant. Whether a discrepancy exists, and whether new physics
is needed to resolve it, depends on details of the systematics of the
object-based methods, and also on the assumptions about other cosmological
parameters and which datasets are combined in the case of the all-sky methods.Comment: Extensively revised and updated since the 2007 version: accepted by
Living Reviews in Relativity as a major (2014) update of LRR 10, 4, 200
Physics, Astrophysics and Cosmology with Gravitational Waves
Gravitational wave detectors are already operating at interesting sensitivity
levels, and they have an upgrade path that should result in secure detections
by 2014. We review the physics of gravitational waves, how they interact with
detectors (bars and interferometers), and how these detectors operate. We study
the most likely sources of gravitational waves and review the data analysis
methods that are used to extract their signals from detector noise. Then we
consider the consequences of gravitational wave detections and observations for
physics, astrophysics, and cosmology.Comment: 137 pages, 16 figures, Published version
<http://www.livingreviews.org/lrr-2009-2
Synthesis and characterization of the tetranuclear iron(III) complex of a new asymmetric multidentate ligand. A structural model for purple acid phosphatases
The ligand, 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H(3)HPBA), which contains a donor atom set that mimics that of the active site of purple acid phosphatase is described. Reaction of H(3)HPBA with iron(III) or iron(II) salts results in formation of the tetranuclear complex, [Fe-4(HPBA)(2)(OAc)(2)(mu-O)(mu-OH)(OH2)(2)]ClO4 center dot 5H(2)O. X-Ray structural analysis reveals the cation consists of four iron(III) ions, two HPBA(3-) ligands, two bridging acetate ligands, a bridging oxide ion and a bridging hydroxide ion. Each binucleating HPBA(3-) ligand coordinates two structurally distinct hexacoordinate iron(III) ions. The two metal ions coordinated to a HPBA(3-) ligand are linked to the two iron(III) metal ions of a second, similar binuclear unit by intramolecular oxide and hydroxide bridging moieties to form a tetramer. The complex has been further characterised by elemental analysis, mass spectrometry, UV-vis and MCD spectroscopy, X- ray crystallography, magnetic susceptibility measurements and variable-temperature Mossbauer spectroscopy
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