Inhibiting epidermal growth factor receptor at a distance.

The epidermal growth factor receptor (EGFR) tyrosine kinase is implicated in a large number of human cancers. Most EGFR inhibitors target the extracellular, growth factor-binding domain or the intracellular, ATP-binding domain. Here we describe molecules that inhibit the kinase activity of EGFR in a new way, by competing with formation of an essential intradimer coiled coil containing the juxtamembrane segment from each member of the receptor partnership. The most potent molecules we describe bind EGFR directly, decrease the proliferation of wild-type and mutant EGFR-dependent cells lines, inhibit phosphorylation of EGFR and downstream targets, and block coiled coil formation as judged by bipartite tetracysteine display. Potency is directly correlated with the ability to block coiled coil formation within full-length EGFR in cells

Rotamer-Restricted Fluorogenicity Of The Bis-Arsenical ReAsH

Fluorogenic dyes such as FlAsH and ReAsH are used widely to localize, monitor, and characterize proteins and their assemblies in live cells. These bis-arsenical dyes can become fluorescent when bound to a protein containing four proximal Cys thiols—a tetracysteine (Cys4) motif. Yet the mechanism by which bis-arsenicals become fluorescent upon binding a Cys4 motif is unknown, and this nescience limits more widespread application of this tool. Here we probe the origins of ReAsH fluorogenicity using both computation and experiment. Our results support a model in which ReAsH fluorescence depends on the relative orientation of the aryl chromophore and the appended arsenic chelate: the fluorescence is rotamer-restricted. Our results do not support a model in which fluorogenicity arises from the relief of ring strain. The calculations identify those As–aryl rotamers that support fluorescence and those that do not and correlate well with prior experiments. The rotamer-restricted model we propose is supported further by biophysical studies: the excited-state fluorescence lifetime of a complex between ReAsH and a protein bearing a high-affinity Cys4 motif is longer than that of ReAsH-EDT2, and the fluorescence intensity of ReAsH-EDT2 increases in solvents of increasing viscosity. By providing a higher resolution view of the structural basis for fluorogenicity, these results provide a clear strategy for the design of more selective bis-arsenicals and better-optimized protein targets, with a concomitant improvement in the ability to characterize previously invisible protein conformational changes and assemblies in live cells

Arsenite binds to the RING finger domains of RNF20-RNF40 histone E3 ubiquitin ligase and inhibits DNA double-strand break repair.

Arsenic is a widespread environmental contaminant. However, the exact molecular mechanisms underlying the carcinogenic effects of arsenic remain incompletely understood. Core histones can be ubiquitinated by RING finger E3 ubiquitin ligases, among which the RNF20-RNF40 heterodimer catalyzes the ubiquitination of histone H2B at lysine 120. This ubiquitination event is important for the formation of open and biochemically accessible chromatin fiber that is conducive for DNA repair. Herein, we found that arsenite could bind directly to the RING finger domains of RNF20 and RNF40 in vitro and in cells, and treatment with arsenite resulted in substantially impaired H2B ubiquitination in multiple cell lines. Exposure to arsenite also diminished the recruitment of BRCA1 and RAD51 to laser-induced DNA double-strand break (DSB) sites, compromised DNA DSB repair in human cells, and rendered cells sensitive toward a radiomimetic agent, neocarzinostatin. Together, the results from the present study revealed, for the first time, that arsenite may exert its carcinogenic effect by targeting cysteine residues in the RING finger domains of histone E3 ubiquitin ligase, thereby altering histone epigenetic mark and compromising DNA DSB repair. Our results also suggest arsenite as a general inhibitor for RING finger E3 ubiquitin ligases

Fish Assemblage of Cedar Fork Creek, Ohio, Unchanged for 28 Years

Author Institution: Dept of Evolution, Ecology & Organismal Biology, The Ohio State University, Mansfield, OHAuthor Institution: Museum of Comparative Zoology, Dept of Ichthyology, Harvard University, Cambridge, MAA 270 m section of Cedar Fork Creek, a clear, gravel-bottomed, headwater stream of the Ohio River System in north-central Ohio, was sampled 48 times from 1975-2003. During the 28 year period 32,237 individuals of 10 fish families and 44 species were collected. Eight taxa made up 86% of the total number collected. Cyprinids (Luxilus cornutus, Campostoma anomalum, Pimephales notatus, Semotilus atromaculatus, and Notropis buccatus) accounted for 65% of the individuals. Two darter species Etheostoma caeruleum, E. nigrum) made up 17.4% of the total, and the white sucker, Catostomus commersoni, accounted for 3.3%. The same common species were abundant throughout the 28 years, and the same rare species were consistently present in small numbers. Species richness averaged 23 species per year. Margalef’s index of diversity varied only slightly from 2.8 to 3.6 during the study indicating the constancy of species composition. Two jackknife estimators (nonparametric resampling procedures) suggested that the collections detected 97-100% of the species present. Exotic species failed to make inroads into the Cedar Fork community except for carp, Cyprinus carpio, that have been in Ohio since 1879, and brown trout, Salmo trutta, that were recently stocked by the Ohio Department of Natural Resources

Nutrients, Eutrophic Response, and Fish Anomalies in the Little Miami River, Ohio

Author Institution: Dept of Biological Sciences, Northern Kentucky University, Highland Heights, KYAuthor Institution: University of Cincinnati, Dept of Biological Sciences, Cincinnati, OHWe documented the eutrophic and chemical environment in the Little Miami River (LMR) to better understand the interaction between eutrophication, eutrophic response variables, and the health of aquatic organisms. Total phosphorus (TP) and soluble reactive phosphorus (SRP), nitrogen, dissolved oxygen (DO), benthic and sestonic algal biomass, benthic phosphorus storage, aqueous trace metals (Cd, Cr, Cu, Se, Zn), heavy metals (Al, Fe, Mn) and major cations (Ca, K, Mg, Na, Si) were analyzed at twelve sites over two summers. Results showed excess TP (>70 ug/L, p <0.05) and SRP (≥62.5 ug/L, p <0.05), borderline nuisance benthic algal biomass (mg/L chlorophyll a/m2) (periphyton: mean = 73.8 +/- 74.2, n = 125; Cladophora: mean = 216.7 +/- 380.7, n = 54), excess benthic phosphorus storage (mg P/m2) (periphyton: mean = 45.5 +/- 23.2, n = 64; Cladophora: mean = 129.3 +/- 224, n = 52), and high daytime DO (mean = 9.1 +/- 1.5 mg/L, n = 132). Previous studies showed aqueous phosphorus concentration and diurnal DO swings were positively correlated with fish anomalies (OEPA 1995, 2000). In this study, however, periphyton phosphorus (P) was the only eutrophic response variable to correlate with the distribution of fish anomalies reported by OEPA in 1995 and 2000, and the association was negative (R2 = 0.143, p = 0.002, m = -1.634, df = 1, 62). We concluded that aqueous nutrients, eutrophic response variables, and/or water chemistry alone did not explain the occurrence of fish anomalies in the LMR

Advances in molecular probe-based labeling tools and their application to multiscale multimodal correlated microscopies

The need to determine the precise subcellular distribution of specific proteins and macromolecular complexes in cells and tissues has been the major driving force behind the development of new molecular-genetic and chemical-labeling approaches applicable to high-resolution, correlated, multidimensional microscopy. This short review is intended to provide an overview of recently developed and widely used electron microscopy (EM)-compatible probes, including tetracysteine tags, mini singlet oxygen generator (MiniSOG), time-specific tag for the age measurement of proteins (TimeSTAMP) with MiniSOG, and enhanced ascorbate peroxidase (APEX). We describe how these highly specific and genetically introduced EM probes are now used, in conjunction with lower resolution light microscopic methods, to obtain wide field or dynamic records of live preparation or of large maps in 3D using recently developed laboratory-scale X-ray microscopes. The article is intended to enable researchers through a high-level view of the toolbox of labels available today for studies aiming to analyze dynamic subcellular and molecular processes in cell culture systems as well as in animal tissues—and ultimately allow investigators to determine the precise location of macromolecular complexes by EM

Potentiometric and conductometric studies of malonyl bis(salicyloylhydrazone) and divalent metal complexes

AbstractA series of complexes of divalent transition metal ions with malonyl bis(salicyloylhydrazone) (H4MSH) have been prepared and characterized with the help of conductometric, potentiometric methods. The proton–ligand and metal–ligand stability constants were obtained pH-metrically. The electrical conductivity of solid complexes was measured at 289K. The low molar conductance values observed for these complexes indicate that, they are non-electrolytes. They are soluble to a limited extent in DMF and DMSO. The elemental analyses of the complexes indicate that the complexes have 1:1 and 2:1 (M:L) stoichiometry with the existence of water, chloride, acetone molecules inside the coordination sphere as evidence from the IR spectral studies. Further, the complexes have been formulated by comparing C, H, N & metal analysis data, and UV–visible spectra of the complexes have been discussed. The protonation constants of the ligand and the stability constants of their metal complexes will be evaluated potentiometrically. The stoichiometric ratios of the complexes formed in solution will be evaluated applying the molar ratio (spectrophotometric) method and confirmed conductometrically